Ligands, including antibodies, showing reactivity against endocrine cells

ABSTRACT

The invention provides monoclonal and polyclonal antibodies recognising molecules on secretory cells of various tissue targets of autoimmune disease allowing a unifying method of preventing and treating autoimmune diseases and other conditions where hormonal dysregulation, hyperinsulinaemia and insulin resistance are involved. It also provides a method for detecting similar antibodies in human sera or other body fluids which can be used in the development of diagnostic kits. Treatment methods arising from this invention comprise the administration of preparations of the antibodies, their target molecules and vectors containing coding sequences of the antibodies and their target molecules.

This application is a U.S. national stage of International ApplicationNo. PCT/GB98/02151 filed Jul. 20. 1998.

FIELD OF INVENTION

This invention describes the development of unique autoantibodies whichare the cause of several autoimmune and other diseases. It providesdiagnostic and prophylactic uses for such antibodies in monoclonal andpolyclonal form and also for the molecules recognised by the antibodies.More specifically, the invention provides for the use of theseantibodies and the molecules they recognise as specific inhibitors ofthe development of autoantibodies with the same specificity in childrenand adults. These antibodies and target molecules are claimed to be ofdiagnostic, prophylactic and treatment use in a wide variety ofautoimmune and other diseases. However, most of the background to theinvention will focus on diabetes, not by way of limitation but by way ofillustration or example.

The Spectrum of Human Autoimmune Diseases

Diseases associated with autoimmune phenomena can be classified within aspectrum ranging from conditions involving destructive lesions of asingle organ or those in which organ or tissue damage is widelydisseminated.

At the organ-specific end of the spectrum, the organs most commonlyaffected are the thyroid, adrenal glands, stomach and islets ofLangerhans (which contain the insulin producing cells) in the pancreas,while at the non-organ specific pole, rheumatological or systemic (e.g.systemic lupus erythematosus) disorders predominate. Autoimmune diseasesare, in rare cases, connected with fulminant viral infections which canalso result in organ destruction. In autoimmunity, the damaging processis slow and sometimes it takes years before the disease becomesmanifest. The following are organ-specific autoimmune diseases whichresult from autoimmune phenomena involving breakdown in immunologicaltolerance to self antigens.

Thyroid. Thyroid autoimmune disease involves a variety of clinicalconditions which result in a common histopathological picture. There isdiffuse infiltration of the gland by mononuclear lymphoid cells. Theconstituent diseases are primary myxoedema, Hashimoto's thyroiditis andGraves' disease. Progression from one to another is not uncommon.Primary myxoedema is the most common form of spontaneous hypothyroidismand is the last stage of the chronic inflammatory process. There is nogoitre formation and the gland is almost completely atrophied.

Hashimoto's thyroiditis is also linked to hypothyroidism but isassociated with goitre. In its various clinical forms, levels of thyroidhormone can either be compensated by increased levels of thyroidstimulating hormone (TSH) produced by the pituitary, or there can beclinical hypothyroidism in spite of raised TSH levels. In bothautoimmune destructive conditions, women are affected five times morefrequently than men.

The most common form of thyrotoxicosis is Graves' disease with orwithout goitre or exophthalmus. It is characterised by remissions andexacerbations. Despite that, the autoimmune process leads tohyperstimulation of the gland, due to the production of thyroidstimulating antibodies, final destruction of the thyroid often occurs.There is a female to male preponderance of 5:1.

In all thyroid autoimmune diseases, the demonstration of variousautoantibodies to the gland confirms clinical diagnosis. Autoantibodiescan be directed against thyroid cytoplasmic antigens, such asthyroglobulin, cell surface components, such as thyroid peroxidase, andthyrocyte surface expressed TSH receptors (i.e. Graves' disease).

Stomach. Autoimmune diseases of the stomach involve either the fundus orthe antrum, leading to various degrees of inflammation affecting thesetwo regions of the gland. In general, this process is named gastritis.In fundal gastritis, there is marked atrophy of the mucosa withconsequent loss of intrinsic factor (IF) production leading tomalabsorption of vitamin B12 and the subsequent development ofpernicious anaemia. In these conditions, antibodies to IF and parietalcells are produced and are present in 90% of affected patients. Parietalcells are destroyed, but chief cells and mucus cells are also destroyed,despite the absence of circulating antibodies to the latter two celltypes. There is a female to male preponderance of 3:1. Autoantibodies togastrin-producing cells in the antrum have been demonstrated in somepatients with antral gastritis. This type of gastritis is associatedwith gastric ulceration and in a proportion of patients, antibodiesstimulating gastric cells have also been demonstrated.

Adrenals, Gonads and Placenta. Autoimmune disease of the adrenal(Addison's disease) is characterised by heavy mononuclear cellinfiltration of the gland, adrenalitis, and the presence ofautoantibodies to adrenal antigens. The symptoms are hyperpigmentation,weakness, fatigue, hypotension, gastro-intestinal symptoms andhypoglycaemia due to adrenal failure. Here again, the disease occursmainly in women. By immunofluorescence the autoantibodies stain thethree layers of the adrenal cortex, but a sub-type can also cross-reactwith analogous steroid-producing cells in the ovary, testis andplacenta. When these latter specificities are present, they correlatewith pre-clinical or clinically overt gonadal failure.

Pituitary. Lymphocytic hypophysitis is a rare autoimmune condition,resulting in hypopituitarism requiring hormone replacement therapy.There is a prevalence in females and the disease presents with a varietyof other organ-specific autoimmune phenomena or associated disorders.Autoantibodies to prolactin-secreting cells can be detected, as well asother organ-specific antibodies in most cases.

Polyendocrine autoimmunity. Patients with organ-specific autoimmunedisease may present with symptoms associated with failure of endocrineor other target organs (e.g. stomach). However, syndromes of multipleaffected organs are not uncommon and autoantibodies to unaffected organsare also detectable in patients suffering with only one organ-specificdisease. Thyroid and gastric autoimmunity are often seen in the sameindividual. Pernicious anaemia, resulting from fundal gastritis, is fivetimes more frequent in patients with thyroid disorders and 30-50% ofpatients with pernicious anaemia also have a history of thyroid disease.

Associations also exist between adrenalitis and thyroiditis andadrenalitis and insulin dependent diabetes mellitus (IDDM). Often casesstart with thyrotoxicosis and Addison's disease simultaneously and manypatients with Addison's disease have at least one other autoimmunedisease. Although hypophysitis and vitiligo (a condition which leads topatchy depigmentation of the skin, most likely due to autoimmunedestruction of resident melanocytes) are rare, they often coexist withother overt organ specific conditions. The serological features, (i.e.the presence of circulating autoantibodies) which overlap among theseautoimmune disorders is far more common than the co-existence of overtdisease. For example, parietal cell antibodies are present in 50% ofpatients with thyroid disorders and 30% of patients with IDDM.

Current Knowledge Regarding the Pathology of IDDM

The current state of knowledge regarding the aetiology of IDDM (type Idiabetes) focuses on the autoimmune destruction of islet β cells in anenvironment of a variety of circulating autoantibodies, autoreactive Tcells in the circulation and in pancreatic islets (insulitis) and avariety of cytokines. A pathogenic role for autoantibodies has thus farnot been demonstrated but their presence has been shown to be ofpredictive value for the identification of preclinical diabetesparticularly in first degree relatives of IDDM patients. Consequentlythe immunological attack resulting in IDDM is considered to be a T celldependent β cell destruction (Tisch and McDevitt; 1996). The evidencewhich lends support to this view is the presence of mononuclear cellinfiltrates (insulitis) in the islets at disease onset (Gepts, 1965;Roep, and DeVries, 1992), the effect of immunosuppressive drugs indelaying disease onset (Bougneres et al., 1988), the destruction ofpancreatic grafts in IDDM patients associated with insulitis (Sibley etal., 1985) and animal studies showing that splenic T-cells are able totransfer diabetes and both CD4 and CD8 T-cells are required (Bendelac elal., 1987). Cloned CD4 T cell lines with specificity for islet cellantigens have also been shown to be diabetogenic (Haskins and McDuffie,1992). There is no evidence to date, however, that T cells cause theinitial damage to islet β cells or any indication as to what the targetantigens might be.

Recently it has been demonstrated that autoreactivity of circulating Tcells to islet cell antigens is not limited to IDDM patients but is alsopresent in healthy, age matched controls even though to a lesser extent.It is therefore very likely that T-cell autoimmune phenomena are aconsequence of β cell dysfunction leading to β cell damage.

Studies examining the cytokine profiles and Th1 and Th2 balances duringdisease progression in NOD mice have demonstrated that Th1 cells and Th1type cytokines predominate at the onset of IDDM. A detailed study toexamine the apparent correlation between the shift in cytokine levelsand IDDM was carried out by Shimada et al. (1996). Splenocytes obtainedfrom NOD and NOD-IA^(k) and BALB/c control mice at various times duringthe disease process were separated to obtain CD45RB low (memory) CD4+ Tcells; these were activated with anti-CD3 and the released cytokinesassayed. A high IFNγ/IL4 ratio was found in NOD mice at, or just before,the onset of hyperglycaemia. The authors proposed that IDDM in the NODmouse progresses as an inflammatory β cell dysfunction without actualcell destruction until late in the disease process.

Dysfunction of β cells indicated by elevated serum proinsulin levelsrelative to insulin or C-peptide has been noted at the clinicalpresentation of juvenile IDDM (Ludvigsson and Heding, 1982). Roder etal. (1994) studied 23 autoantibody positive siblings of IDDM patients,who were divided to 2 groups according to their first phase insulinresponse (FPIR) to intravenous glucose. Eleven siblings had diminishedFPIRs and their fasting proinsulin/insulin or C-peptide ratios were 2-3fold higher than the remainder of the siblings who had normal FPIRs.Nine of the 11 siblings with low FPIRs and high proinsulin/insulin orratios became diabetic 1-28 months after testing, compared to none amongthe remainder of the siblings.

Dysfunction and eventual destruction of β cells is characteristic ofIDDM, however, both recent onset and long standing type 1 diabetics alsohave defective glucagon and epinephrine secretory responses and hepaticglucose production during hypoglycaemia (Kleinbaum el al., 1983).Carefully controlled studies using a hyperinsulinaemic hypoglycaemicclamp technique have demonstrated that glucagon levels rise during a 180minute insulin infusion in normal but not in diabetic subjects (Barrouet al., 1994). Hepatic glucose production is also severely impaired inthe latter group. This defect in counterregulation of hypoglycaemia isrefractory to intensive medical management in many type 1 diabetics.

Although it is clear that glucagon secretion is impaired in longstanding diabetics, not much is known about glucagon secretion inprediabetic individuals. Abnormalities in glucagon secretion have beendemonstrated in animal models of diabetes. An investigation inprediabetic and overtly diabetic NOD mice revealed that in theprediabetic animals when fasting blood glucose and plasma insulin levelswere normal, plasma glucagon levels were markedly elevated compared tocontrol mice (Ohneda et al., 1984). Therefore an underlying metabolicdisorder existed before the onset of diabetes.

Glucagon secreted from the pancreatic α cells is an important factor inmaintaining normal control of euglycaemia by stimulating hepatic glucoseproduction and also potentiating glucose induced insulin secretion. Thiswas demonstrated by a drop in insulin secretion afterimmunoneutralisation of glucagon in fasted rats which was notaccompanied by a drop in blood glucose (Brand et al., 1995).

Insulin release from FACS separated single β cells is poor in responseto nutrient challenge. This secretory defect can be fully restored byrecombining the β cells with separated α cells or by addition of (Bu)₂cAMP or glucagon (Pipeleers et al., 1985). Glucagon is the major factorin elevating cAMP levels in β cells (Rasmussen et al., 1990). Isolated βcells exhibit lower levels of cAMP than β cells in intact islets;increased or decreased c AMP levels in islets are paralleled by risingor falling secretory responses to glucose (Howell et al., 1973). ThecAMP levels can be restored by reaggregation with non-β islet cells oraddition of glucagon (Schuit and Pipeleers, 1985). Compatible with adirect effect of glucagon on β cells is the demonstration of glucagonreceptors on β cells (Van Schravendijk et al., 1985). Glucagon has alsobeen shown to enhance the amplitude of pulsatile insulin release inresponse to glucose without affecting the periodicity of the secretorypulses (Marchetti et al., 1994).

Since it is well established that a rise in cytoplasmic Ca²⁺concentration is essential for insulin secretion (Prentki andMatschinsky, 1987), glucagon induced rises in cAMP levels have beenproposed to act via increased Ca²⁺ levels. Intricate experimentsmeasuring Ca²⁺ transients under the effect of photoreleasedintracellular cAMP from a caged precursor demonstrated that the increasein Ca²⁺ transients accounted for 10% of the total increase in exocytosisproduced by cAMP (Ammala, et al., 1993). By similar methods Ammala etal., (1993) also demonstrated that cAMP initiates exocytosis at a Ca²⁺concentration which by itself is unable to promote secretion and alsoenhances exocytosis at higher Ca²⁺ concentrations. Westerlund et al.(1997) also demonstrated that insulin secretion continued to bepulsatile under conditions when [Ca²⁺]i remained stable. Theseexperiments demonstrate that cAMP sets the threshold of sensitivity forthe secretory action of Ca²⁺ channel activation, whereby the role ofglucagon (in increasing cAMP levels in β cells) becomes of primeimportance in controlling the amplitude of glucose induced fast insulinsecretory pulses.

Glucagon secretion is also pulsatile, Storch et al., (1993) reportedthat the plasma concentration of glucagon in liver cirrhosis patientsvaried considerably in intervals of 4.1-6.5 minutes. In vitro perfusedrat pancreata secreted both insulin and glucagon in pulses of 5.8 and6.5 minutes respectively; reversing the direction of perfusion in ratand dog pancreata did not affect the periodicity of hormone secretion(Stagner and Samols, 1988). This negates the possibility that directintra-islet hormonal interactions or a venous hormone sensitive receptormechanism is responsible for the periodicity of secretion. Single mouseislets secrete insulin in response to glucose stimulation by both slowand fast oscillatory pulses (Bergsten et al., 1994). The mechanismtherefore for pulsatile insulin secretion resides within individualislets; this is distinct from [Ca²⁺]i transients demonstrated byexperiments involving activation of protein kinase C which increased theamplitude of oscillations without affecting their frequency or changesin [Ca²⁺]i (Deeney et al., 1996). These reports demonstrate that thepacemaker for pulsatility of insulin and glucagon secretion is locatedwithin the islets and is independent of extrinsic innervation and directhormonal interaction. This has also been established in man bydemonstration of both low and high frequency pulsatilities in successfulpancreas transplants (Sonnenberg et al., 1992).

The Role of the Pacemaker in NIDDM

Beta-cell dysfunction is prominent in type 2 non-insulin-dependentdiabetes (NIDDM) which is a disease also involving insulin resistance.The relative importance of these two components has been controversial.Early on in the disease there is a marked disruption in pulsatileinsulin secretion with loss of the high frequency pulses and a reductionin amplitude of slow oscillations (Leahy, 1990; Guillausseau, 1994). Theloss of pulsatile secretion may be an important potential contributor toinsulin resistance. Various studies designed to identify predictors forthe development of NIDDM have concluded that β cell dysfunction ratherthan insulin resistance is the major factor predisposing to NIDDM(Pimenta et al., 1995; Davis et al., 1995; Nijpels et al., 1996).Therefore the cause of NIDDM must be related to the event that inducesthe dysfunction. It is proposed herein that the dysfunction isconsequent to the disruption of the pacemaker which maintains pulsatilesecretion of both insulin and glucagon.

Parksen et al. (1995) have examined the contribution of pulsatile vs.basal insulin secretion in the overnight-fasted dog and havedemonstrated that the majority of insulin (70%) was secreted as pulses.Disruption of this system would therefore have a major impact on totalinsulin secretion.

The natural history of β cell dysfunction preceding IDDM is moredifficult to study because of the abrupt and destructive nature of thedisease at diagnosis. O'Meara et al., (1995) were, however, able tostudy an individual over a 13-month period leading to the development ofIDDM. When fasting glucose and glycosylated haemoglobin concentrationswere still within normal range, insulin responses to intravenous glucosewere reduced. The oscillatory pattern of secretion was preserved but thesecretory responses were reduced.

DESCRIPTION OF THE INVENTION

The invention relates to a new concept regarding the cause of autoimmunediseases and specifically describes its application to types 1 and 2diabetes as an illustration and not as a limitation. The presentinvention provides monoclonal or polyclonal antibodies or functionallyequivalent ligands with reactivity against an anti-TCR Vβ antibody, foruse as a pharmaceutical or as a diagnostic agent. These molecules mayalso exhibit reactivity against GPI-linked TCR Vβ chains, phospholipids,phospholipid glycans, single stranded DNA and/or double stranded DNA.The invention also provides the use of these antibodies in themanufacture of a medicament for the treatment of IDDM, NIDDM, or organor non-organ specific autoimmune and related diseases. Preferably, amonoclonal antibody is used in accordance with the present invention.

With regard to diabetes, the invention implicates dysregulation of αcell function by newly identified autoantibodies with similarspecificity as the said monoclonal antibodies as the major factor in thediabetogenic process. To substantiate this new concept, the inventiondemonstrates that the said monoclonal antibodies recognise a commonepitope on a set of signalling molecules on α cells (which may functionas the pacemaker in the islets) which are the targets of the saidpathogenic autoantibodies.

The invention also provides for the detection of the said autoantibodiesand furthermore embodies the use of these and the said MoAbs and themolecules recognised by these MoAbs in prophylactic and therapeuticinterventions of autoimminue and related diseases including IDDM andNIDDM.

The monoclonal antibodies dysregulated insulin secretion from humanislet cell cultures (see experimental section for details and Table 1).They were localised to α cells by simultaneously staining pancreaticsections with the said MoAbs (IgM) and also anti-glucagon MoAbs (IgG),detecting binding with fluoresceinated anti-mouse IgM and rhodaminatedanti-mouse IgG respectively; the staining patterns of the antibodieswere identical, demonstrating that both were staining the same glucagonproducing α cells.

It is thought that the affect of the pathogenic autoantibodies on the αcell causes loss of both glucose counterregulatory responsiveness andthe fine-tuning of insulin secretion. The dysregulation of insulinsecretion results either in β cell death leading to IDDM or continuedsurvival of the β cell in the dysregulated state leading to NIDDM. Thesetwo outcomes are dependent upon the genetic susceptibility of theindividual. In IDDM, T cell sensitisation is secondary to β cell damageand may accelerate the death of remaining β cells. However, theapplicant does not wish to be bound by this theory.

The MoAbs which identified the α cell surface molecules were raised byimmunising mice with anti-TCR Vβ monoclonal antibodies, as describedbelow in the experimental section. Monoclonal antibodies produced by theresulting clones either recognised the anti-Vβ immunogen alone, orrecognised the immunogen as well as phospatidyl inositol, phosphatidylserine, cardiolipin (diacyl glycerol) and ds and ss DNA. These lattermonoclonals also recognised human pancreatic α cells (FIG. 1),follicular cells of the thyroid (FIG. 2), cells of the adrenal medulla(FIG. 3), stomach and intestinal tract (FIG. 4), stomach, salivaryglands, ovary, striated muscle, connective tissue, stated herein by wayof example and not limitation.

As used herein, the term “functional equivalent” is intended to describecompounds that possess the desired binding site and includes anymacromolecule or molecular entity that binds an anti-TCR Vβ antibodywith a dissociation constant of 10⁻⁴M or less, preferably 10⁻⁷M or less,most preferably 10⁻⁹M or less, and that possesses an equivalentcomplementarity of shape to that possessed by the binding sites of theanti anti-TCR Vβ antibodies identified herein.

Current methods of generation of compounds with affinity for a moleculeof interest have been until recently relatively primitive. The notion ofcombinatorial chemistry and the generation of combinatorial librarieshas, however, developed at great speed and facilitated the rationaldesign and improvement of molecules with desired properties. Thesetechniques can be used to generate molecules possessing binding sitesidentical or similar to those of the antibodies identified herein.

Such compounds may be generated by rational design, using for examplestandard synthesis techniques in combination with molecular modellingand computer visualisation programs. Under these techniques, the “lead”compound with a similar framework to the antibody binding site isoptimised by combining a diversity of scaffolds and componentsubstituents.

Alternatively, or as one step in the structure-guided design of amolecular entity, combinatorial chemistry may be used to generate orrefine the structure of compounds that mimic the relevant binding siteby the production of congeneric combinatorial arrays around a frameworkscaffold. These steps might include standard peptide or organic moleculesynthesis with a solid-phase split and recombine process or parallelcombinatorial unit synthesis using either solid phase or solutiontechniques (see, for example Hogan, 1997 and the references citedtherein).

Alternatively, or as a portion of a molecule according to this aspect ofthe present invention, functional equivalents may comprise fragments orvariants of the identified antibodies or closely related proteinsexhibiting significant sequence homology. By fragments is meant anyportion of the entire protein sequence that retains the ability to bindto an anti-TCR Vβ antibody with a dissociation constant of 10⁻⁴M orless, preferably 10⁻⁷M or less, most preferably 10⁻⁹M or less.Accordingly, fragments containing single or multiple amino aciddeletions from either terminus of the protein or from internal stretchesof the primary amino acid sequence form one aspect of the presentinvention. Variants may include, for example, mutants containing aminoacid substitutions, insertions or deletions from the wild type sequenceof the antibody.

Biologically-active peptides with binding sites that mimic theantibodies described herein may be generated using phage libraries.Nucleic acids encoding amino acid residues identified as participants inthe binding site, together with nucleic acid encoding the surroundingframework residues may be fused to give a polypeptide unit of between 10and 1000 residues, preferably between 25 and 100 residues. By fusion ofthis nucleic acid fragment with that encoding a phage protein, forexample pIII of the bacteriophage fd, the fusion molecule may bedisplayed on the surface of phage. Screening of the phage library withanti-TCR Vβ antibody will then identify those clones of interest. Theseclones can then be subjected to iterative rounds of mutagenesis andscreening to improve the affinity of the generated molecules for theirtarget.

The antibodies or functionally equivalent ligands according to thepresent invention may be of vertebrate or invertebrate origin.Preferably, the antibodies are derived from B cells immortalised byEpstein-Barr virus transformation or other methods using B cellsobtained from healthy or diseased humans or animals.

The antibody or equivalent ligand may be isolated by passing body fluidfrom animals or humans down an antigen-conjugated column. The animalsmay have previously been immunised with antigen, may be diseased or mayhave been manipulated by drug or by diet so as to develop a disease.

According to a still further aspect of the invention, there is provideda peptide, oligopeptide, polypeptide or protein that is bound by themonoclonal or polyclonal antibody or equivalent ligand according to thefirst aspect of the invention, which is not an anti-TCR Vβ antibody, foruse as a pharmaceutical or as a diagnostic agent. Of particularpreference for use in this aspect of the present invention are proteinsencoded by clones 1.1, 1.2, 1.3, 3.1, 4.1, 5.1, 5.2 or 5.3 as describedbelow, fragments thereof and functional equivalents thereof. Suchmolecules may also be used in the manufacture of a medicament for thetreatment of IDDM, NIDDM, or other organ or non-organ specificautoimmune and related diseases.

These molecules are recognised by the anti-anti-Vβ monoclonal antibodiesand were identified by screening a human pancreas cDNA λgt11 library.Eight cDNA clones were purified and sequenced. Clones 1.1, 1.2 and 1.3code for a secretogranin 1 like protein: clones 3.1, 4.1 and 5.1 codedfor a 67 kd laminin receptor like protein; clone 5.2 coded for a newmolecule that the inventor has named ESRP1 (endocrine secretionregulatory protein 1). Clone 5.3 codes for a human zymogen granule GP-2protein-like protein.

The unifying characteristics of all these molecules is that they arelinked to the cell membrane via a novel glycosyl phosphatidyl inositol(GPI) anchor. The regulation and expression of GPI-linked molecules oncell surfaces has been described (Low, 1989; Udenfriend and Kodukula,1995). These acyl residues are sensitive to insulin action via insulinactivated phospholipases (Chan et al. 1988). The cleavage products ofthese molecules are internalised by the α cells and are postulatedherein to regulate glucagon secretion. The molecules require time to beresynthesised and reexpressed on the membrane which accounts for theperiodicity of glucagon secretion and thus pulsatile secretion of bothglucagon and insulin. This type of mechanism can account for thepacemaker in the islets. Antibodies which bind to the region on thesemolecules that undergo enzymatic cleavage can interfere with the actionof the enzyme and thereby disrupt the regulation of glucagon and insulinsecretion.

There are various mechanisms by which antibodies with similarspecificity may arise physiologically in humans. Firstly, environmentalagents such as infections or superantigens can induce clonal expansionof T cells and during this proliferative phase abnormally developedpartial TCR complexes are retained intracellularly and degraded. T cellscan apoptose under certain conditions and release degraded TCR productswhich can be immunogenic and trigger the cascade of anti-Vβ andanti-anti-Vβ network of antibody production. Secondly, it has beenreported by Bell et al., (1994) that a signal peptide for a GPI anchorattachment is present in the TCRβ chain polypeptide sequence and thatTCRβ chains lacking the cytoplasmic tail sequence are expressed on amature T cell hybridoma line as a GPI-anchored monomeric polypeptide inthe absence of TCRα. GPI-linked TCRβ chains have been detected in TCRβtransgenic mice but not in normal mice: therefore the abnormalexpression of such Vβ chains can induce a cascade of network responsesresulting in antibodies of similar specificity to anti-anti-Vβ reagents.Such antibodies were detected in human sera in another embodiment ofthis invention (see Table 2 in experimental section).

The cDNA clones mentioned above and the proteins that they encode aredescribed in further detail below:

Clones 1.1, 1.2, 1.3. These three clones 1500 bp, 1400 bp and 900 bprespectively code for a Secretogranin 1 (Sg1) like molecule.Secretogranin 1 is a 657 amino acid long polypeptide of 76 kd and ispreceded by a cleaved N-terminal signal peptide of 20 residues (Benedumet al., 1987). It has a disulfide bonded loop structure and is asecretory protein sorted to secretory granules of endocrine cells andneurones. It was demonstrated by Pimplikar and Huttner, (1992) that inthe neuroendocrine cell line PC12, a fraction of exocytosed Sg1 was notreleased but remained associated with the plasma membrane. The surfaceSg1 (approximately 10% of the total cellular protein) was internalisedand degraded indicating possible signalling properties. This polypeptidehas the characteristics of a caveolar protein (Chang el al., 1994). Thepromoter region of the mouse Sg1 gene contains a cAMP-responsive element(Pohl et al., 1990). Secretogranins are a family of acidic proteins.Because they are not found in exocrine cells they have been used asimmunohistochemical diagnostic markers for endocrine tumours. Inaddition Sg1 is a heparin-binding adhesive protein and has been shown tomediate substratum adhesion (Chen el al., 1992). It is of interest thatin the rodent Sg1 mRNA accumululation begins around embryonic days 13-14and peaks by postnatal day 20 (Foss-Peters et al., 1989).

Clones 3.1, 4.1, 5.1. Three other clones 3.1(900 bp), 4.1(900 bp) and5.1(1000 bp) code for a 67 kd laminin receptor like protein. Laminin isa major component of basement membranes which plays an important role ina variety of cell functions such as adhesion, tissue remodelling, woundhealing, inflammation, tumour cell metastasis etc. Interaction of theextracellular matrix (ECM) with cells in contact with it is via distinctcell surface receptors such as the 67 kd laminin receptor. This lamininbinding protein also binds elastin, collagen type IV and is agalactolectin which enables its purification on glycoconjugate affinitycolumns containing β galactosugars. Beta galactose sugars such asgalactose and lactose can elute this protein from elastin or lamininaffinity columns (Hinek, 1994). The binding of galactosugars to thelectin site on the molecule not only has the effect of displacing theECM ligands from their binding site but also dissociates the 67 kdprotein from the cell membrane (Hinek et al., 1992). It has also beendemonstrated that there is a relationship between deficiency of thisprotein on smooth muscle cells from ductus arteriosus, their detachmentfrom elastin and their capability of migration.

The surface expression of this protein is likely to be undertranslational regulation. Transfected cells which expressed high levelsof mRNA did not always express correspondingly high levels of theprotein on their surface. (Landowski, et al., 1995).Microspectrofluorometry and videomicroscopy experiments havedemonstrated that the binding of elastin or the active peptide VGVAPG(SEQ ID NO:7) to aortic smooth muscle results in a transient increase infree intracellular Ca²⁺. This suggests that cell surface laminin orelastin binding protein acts as a true receptor mediating intracellularsignalling (Hinek, 1994).

There is still controversy regarding the mode of attachment of thisprotein to the cell membrane as analysis of the predicted amino acidsequence has not revealed any hydrophobic domains characteristic of atransmembrane region. Methyl esterification of the purified proteinfollowed by gas chromatography and mass spectrometry has indicated thatthe protein is acylated by covalently bound fatty acids, palmitate,stearate and oleate but the linkage chemistry has not been definitivelyidentified (Landowski et al., 1995). Acylation of this protein conferson it a further set of properties apart from those dependent uponlaminin binding. Lipid modifications have been shown to affectprotein-protein interactions and acyl modifying groups may also generatesecond messengers in signal transduction.

Clone 5.2. The polypeptide encoded by this approximately 1200 bp cDNAclone has no significant similarity to a functionally characterisedprotein. It is therefore not possible to obtain any functionalcomparisons with any known proteins. It is thought, however, that thisprotein shares an epitope with the proteins identified by the monoclonalanti-anti-Vβ used in screening the pancreas library and therefore sharessimilar functional properties. This protein will henceforth be calledendocrine secretion regulatory protein 1 (ESRP1).

According to a further embodiment of the invention, there is providedthe protein ESRP1, fragments thereof and functional equivalents. Thesequence of the protein is provided in FIG. 7 below. The sequence of theencoding nucleic acid forms a further aspect of this embodiment of theinvention and is provided in FIG. 6 below.

According to a further aspect of the present invention there is providedthe ESRP1 protein for use in therapy or diagnosis.

An ESRP1 protein or functional equivalent according to the presentinvention may be derived from any organism possessing a protein in thesame family as the compounds identified herein. By protein family ismeant a group of polypeptides that share a common function and exhibitcommon sequence homology between motifs present in the polypeptidesequences.

Preferably, the protein, protein fragment or functional equivalent isderived from a mammal, preferably the human.

According to a still further aspect of this embodiment of the presentinvention there is provided the use of the ESRP1 protein, fragmentsthereof and functional equivalents in the manufacture of a medicamentfor the treatment of IDDM, NIDDM, organ or non-organ specific autoimmunedisease, cardiovascular disease, cancer cachexia and cancer and anyother diseases where anti-phospholipid antibodies and/orhyperinsulinaemia and insulin resistance are present.

As used throughout this specification, the term “organ or non-organspecific autoimmune disease” is meant to include IDDM, NIDDM, autoimmunediseases of the thyroid, adrenal gland, gonads, stomach and pituitary,systemic lupus erythematosus, systemic sclerosis and Sjogren's syndrome.“Cardiovascular disease” is meant to include coronary and carotid arterydisease, macro and micro-vascular angina, peripheral vascular disease,atherosclerosis and hypertension. “Cancer” is meant to include breast,colorectal, gastric, endometrial, prostate, head and neck, lungsarcomas, “Other suitable disease” is meant to include polycystic ovarysyndrome, obesity, Cushing's syndrome and metabolic syndrome X. Thesediseases are given as examples and not as limitations.

Clone 5.3. This approximately 2000 bp cDNA clone codes for protein thatis very similar to the exocrine human zymogen granule membrane GP-2protein. However, clone 5.3 has several nucleic acid and consequentlyamino acid differences and is located in the endocrine pancreas.

Since the antibody reactive with this cDNA clone stains the endocrinepancreas, the protein it codes for is thought to be the endocrinecounterpart of the exocrine GP-2 protein. This does not, however, conferupon this protein the same function in the endocrine cells as it has inthe exocrine tissue. Rat GP-2 expressed in cell lines of endocrine orexocrine origin by cDNA transfection, was shown to be targeted tosecretory granules in the exocrine cells but not in the endocrine (Hoopset al., 1993).

The major protein in isolated zymogen granule membranes of the exocrinepancreas is GP-2 which accounts for up to 40% of the total protein(Ronzio et al., 1978). Both the human and rat proteins are attached tothe granule membrane via a glycosyl phosphatidyl inositol (GPI) linkageand can be released from the membrane by phosphatidyl inositol-specificphospholipase C (PI-PLC). The high content of GP-2 in zymogen granulemembranes has led to the hypothesis that this protein is important ingranule formation. It has, however, been reported that GP-2 mRNA isabsent from the embryonic rat pancreas and GP-2 is expressed only afterbirth during the period of weaning. Since the embryonic rat pancreascontains plenty of granules it can be inferred that GP-2 is notessential for granule formation (Dittie and Kern, 1992). Theseobservations have been confirmed in studies of the pig pancreas wherethe GP-2 protein and mRNA are also absent in the foetus and only startbeing produced 21 days after birth. Foetal granules are thereforecompletely devoid of GP-2 protein (Laine el al., 1996). The emergence ofantigens at the time of weaning could explain the concomitantdevelopment of insulitis at this time in experimental animal models ofdiabetes.

The precise functional role/s of GP-2 protein are not known but sincethe protein exists both in soluble form (40%) and membrane bound (60%)in the zymogen granules it must have both intracellular andextracellular functions. Since the GP-2 protein is expressed after birthin rodents and pigs, tolerance to this molecule must be peripherallyinduced rather than intrathymically during embryonic development.Pulendran et al. (1995) and Shokat and Goodnow (1995) have demonstratedthat germinal centre B cells become apoptotic upon encountering solubleantigen. Therefore soluble GP-2 may have the role of inducing toleranceby binding to the immunoglobulin receptor of GP-2 reactive germinalcentre B cells and triggering apoptosis.

The membrane bound form of GP-2 can be released from the membrane byproteases and phospholipases; the presence of inositol 1,2-(cyclic)monophosphate on secreted hydrophilic GP-2 has been demonstratedconfirming the action of a phospholipase C in the cleaving of GP-2 fromthe membrane (Paul el al., 1991). The lipid products such as1,2-diacylglycerol and phosphatidic acid or inositol glycan derived fromlipid anchors of cell surface proteins by phospholipases, proteases orhydrolases may be internalised and participate in second messengerpathways. GPI-linked proteins may also be directly involved in signaltransduction via crosslinking of their NH₂ terminal domains. The signaltransduction is via the src family protein tyrosine kinases p56 lck andp59 fyn and involves the GPI anchor (Shenoy-Scaria et al., 1992). GP-2protein has also been shown to have enzymatic properties and has beenidentified as a nucleoside phosphatase with di- and tri-phosphataseactivities within the zymogen granule membrane of the pig. This impliesthat it is involved in energy-requiring processes in the cytosol(Soriani and Freiburghaus, 1996).

The GP-2 protein has 53% identity and 85% similarity to the humanUromodulin/Tamm-Horsfall (THP) protein over a 450 amino acid stretch atthe C-terminal region. THP is also GPI-linked and both proteins belongto a family of proteins including the sperm receptors Zp2 and Zp3 and βglycan (TGF-β type III receptor) and are characterised by a 260 residuedomain common to these apparently diverse proteins (Bork and Sander,1992). The newly identified α cell protein encoded by the 2000 bp cDNAclone must also belong to this family of proteins and must have asignificant function in the control of the secretory process of α cells.

For many applications, an antibody or equivalent ligand according to thefirst aspect of the present invention or peptide, oligopeptide,polypeptide or protein recognised by such an antibody may be fused to aneffector or reporter molecule such as a label, toxin or bioactivemolecule. According to a further aspect of the invention there isprovided an antibody or equivalent ligand according to the first aspectof the invention or a peptide, oligopeptide, polypeptide or proteinrecognised by such an antibody that is chemically-modified, bound to abiological or synthetic substance, or conjugated to an enzyme, anindicator compound, a drug, a toxin or a radioactive label, for use as apharmaceutical or as a diagnostic agent.

Suitable labels will be well known to those of skill in the art. Forexample, such labels may comprise an additional protein or polypeptidefused to an antibody, fragment thereof, or equivalent ligand at itsamino- or carboxy-terminus or added internally. The purpose of theadditional polypeptide may be to aid detection, expression, separationor purification of the antibody, fragment thereof, or equivalent ligandor may be to imbue additional properties to the antibody, fragmentthereof, or equivalent ligand as desired.

Particularly suitable candidates for fusion will be reporter moleculessuch as luciferase, green fluorescent protein, or horse radishperoxidase. Labels of choice may be radiolabels or molecules that aredetectable spectroscopically, for example fluorescent or phosphorescentchemical groups. Linker molecules such as streptavidin or biotin mayalso be used. Additionally, other peptides or polypeptides may be usedas fusion candidates. Suitable peptides may be, for example,β-galactosidase, glutathione-S-transferase, luciferase, polyhistidinetags, secretion signal peptides, the Fc region of an antibody, the FLAGpeptide, cellulose binding domains, calmodulin and the maltose bindingprotein.

These fusion molecules may be fused chemically, using methods such aschemical cross-linking. Suitable methods will be well known to those ofskill in the art and may comprise for example, cross-linking of thethiol groups of cysteine residues or cross-linking using formaldehydes.Chemical cross-linking will in most instances be used to fusenon-protein compounds, such as cyclic peptides and labels.

When it is desired to fuse two or more protein molecules, the method ofchoice will often be to fuse the molecules genetically. In order togenerate a recombinant fusion protein, the genes or gene portions thatencode the proteins or protein fragments of interest are engineered soas to form one contiguous gene arranged so that the codons of the twogene sequences are transcribed in frame.

The compounds of the present invention may also be bound to a supportthat can be used to remove, isolate or extract anti-anti-TCR Vβantibodies from body tissues. The support may comprise any suitablyinert material and includes gels, magnetic and other beads,microspheres, binding columns and resins.

Protein or peptide compounds according to the invention will preferablybe expressed in recombinant form by expression of the encoding DNA in anexpression, vector in a host cell. Such expression methods are wellknown to those of skill in the art and many are described in detail inDNA cloning: a practical approach, Volume II: Expression systems, editedby D. M. Glover (IRL Press, 1995) or in DNA cloning: a practicalapproach, Volume IV: Mammalian systems, edited by D. M. Glover (IRLPress, 1995). Protein compounds may also be prepared using the knowntechniques of genetic engineering such as site-directed or randommutagenesis as described, for example, in Molecular Cloning: aLaboratory Manual: 2nd edition, (Sambrook el al., 1989, Cold SpringHarbor Laboratory Press) or in Protein Engineering: A practical approach(edited by A. R. Rees et al., IRL Press 1993).

Suitable expression vectors can be chosen for the host of choice. Thevector may contain a recombinant DNA molecule encoding compounds of thepresent invention operatively linked to an expression control sequence,or a recombinant DNA cloning vehicle or vector containing such arecombinant DNA molecule under the control of a promoter recognised bythe host transcription machinery.

Suitable hosts include commonly used prokaryotic species, such as E.coli, or eukaryotic yeasts that can be made to express high levels ofrecombinant proteins and that can easily be grown in large quantities.Mammalian cell lines grown in vitro are also suitable, particularly whenusing virus-driven expression systems such as the baculovirus expressionsystem which involves the use of insect cells as hosts. Compounds mayalso be expressed in vivo, for example in insect larvae or in mammaliantissues.

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising a monoclonal or polyclonalantibody or functionally equivalent ligand with reactivity against ananti-TCR Vβ antibody or a peptide, oligopeptide, polypeptide or proteinrecognised by such antibodies (which is not an anti-TCR Vβ antibody), inconjunction with a pharmaceutically-acceptable excipient. Suitableexcipients will be well known to those of skill in the art and may, forexample, comprise a phosphate-buffered saline (0.001M phosphate salts.0.138M NaCl, 0.0027M KCl, pH7.4). Pharmaceutical compositions may alsocontain additional preservatives to ensure a long shelf life in storage.

The monoclonal or polyclonal antibody or functionally equivalent ligandwith reactivity against an anti-TCR Vβ antibody or peptide,oligopeptide, polypeptide or protein recognised by such an antibody mayconstitute the sole active component of the composition or can form partof a therapeutic package for topical (such as a component of a cream),oral or parenteral administration.

According to a further aspect of the present invention there is providedthe use of an antibody or equivalent ligand with reactivity against ananti-TCR Vβ antibody in the manufacture of a medicament for thetreatment of IDDM, NIDDM, or other organ or non-organ specificautoimmune disease, cardiovascular disease, cancer cachexia and canceror any other diseases where anti-phospholipid antibodies and/orhyperinsulinaemia and insulin resistance are present.

According to a still further aspect of the present invention there isprovided a method of treatment of IDDM, NIDDM, or other organ ornon-organ specific autoimmune disease, cardiovascular disease, cancercachexia and cancer or any other diseases where anti-phospholipidantibodies and/or hyperinsulinaemia and insulin resistance are present.

According to a still further aspect of the present invention there isprovided the use of a peptide, oligopeptide, polypeptide or protein thatis bound by a monoclonal or polyclonal antibody or equivalent ligandwith reactivity against an anti-TCR Vβ antibody, which is not ananti-TCR Vβ antibody, in the manufacture of a medicament for thetreatment of IDDM, NIDDM, or other organ or non-organ specificautoimmune disease, cardiovascular disease, cancer cachexia and canceror any other diseases where anti-phospholipid antibodies and/orhyperinsulinaemia and insulin resistance are present.

According to a further embodiment of the invention, there is provided amethod for the detection of a naturally-occurring autoantibody,comprising contacting a blood, plasma or serum sample or other bodyfluid with a monoclonal or polyclonal antibody or equivalent ligandaccording to the first aspect of the invention and with target moleculesand assessing the amount of said naturally-occurring autoantibody thatbinds specifically to the target molecules. The monoclonal or polyclonalantibody or equivalent ligand may be labelled, for example with anenzyme, so that the labelled antibody or equivalent ligand competes withthe autoantibodies for the target molecules to form complexes. Theamount of label bound in said complexes is thereby inverselyproportional to the concentration of autoantibodies present in saidsample. If labelled with an enzyme, the formation of the complexes willinhibit or inactivate the activity of the enzyme so that the degree ofinhibition or activation is inversely proportional to the concentrationof autoantibodies that are present in the sample.

In one aspect of this embodiment of the invention, the target molecule,which may be for example an anti-TCR Vβ polyclonal or monoclonalimmunoglobulin molecule or any part thereof that identifies at least oneepitope on T cell receptor Vβ chains in humans or any other animalspecies, is bound to an enzyme linked to a substrate such that bindingof antibody to the target molecules activates the enzyme and causes acolour change that is measurable spectrophotometrically. The targetmolecules may be bound to an enzyme that is linked to the substrate andmay be present on a dipstick which can be contacted with said sample.

The invention also comprises the use of an antibody or equivalent ligandwith reactivity against an anti-TCR Vβ antibody or a peptide,oligopeptide, polypeptide or protein that is bound by a monoclonal orpolyclonal antibody or equivalent ligand with reactivity against ananti-TCR Vβ antibody (for example ESRP1) as a component in a kit for thedetection or quantification of levels of naturally-occurringautoantibodies in a patient. Such a kit will resemble a radioimmunoassayor ELISA kit and would additionally comprise detection means that allowsthe accurate quantification of the compound of interest. Such methodswill be apparent to those of skill in the art.

The antibody or equivalent ligand or peptide, oligopeptide, polypeptideor protein that is bound by the monoclonal or polyclonal antibody orequivalent ligand may be bound to magnetic beads, agarose beads or maybe fixed to the bottom of a multiwell plate. This will allow the removalof the unbound compounds from the sample after incubation. Alternativelythe protein may be bound to SPA (Scintillation Proximity Assay) beads,in which case there is no need to remove unbound ligand. Using a set ofunlabelled standards, the results obtained with these standards can becompared with the results obtained with the sample to be measured.

The antibody or equivalent ligand with reactivity against an anti-TCR Vβantibody or peptide, oligopeptide, polypeptide or protein that is boundby a monoclonal or polyclonal antibody or equivalent ligand withreactivity against an anti-TCR Vβ antibody can also be used for thedetection of naturally-occurring autoantibodies in tissue from apatient. Any technique common to the art may be used in such a detectionmethod and may comprise the use of blotting techniques (Towbin et al.,1979), binding columns, gel retardation, chromatography, or any of theother suitable methods that are widely used in the art.

The invention also provides a cDNA, RNA or genomic DNA sequencecomprising a sequence encoding an antibody or equivalent ligandaccording to the first aspect of the invention or encoding a peptide,oligopeptide, polypeptide or protein according to the second aspect ofthe invention, for use as a pharmaceutical or as a diagnostic agent.

With regard to the protein ESRP1, the preferred nucleic acid moleculecomprises a nucleotide fragment identical to or complementary to anyportion of the nucleotide sequence shown in the accompanying FIG. 6 or asequence which is degenerate or substantially homologous therewith, orwhich hybridises with the said sequence. By ‘substantially homologous’is meant sequences displaying at least 50% sequence homology, preferably60% sequence homology. ‘Hybridising sequences’ included within the scopeof the invention are those binding under standard non-stringentconditions (6×SSC/50% formamide at room temperature) and washed underconditions of low stringency (2×SSC, room temperature, or 2×SSC, 42° C.)or preferably under standard conditions of higher stringency, e.g.0.1×SSC, 65° C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).

A nucleic acid sequence according to the invention may be single- ordouble-stranded DNA, cDNA or RNA. Preferably, the nucleic acid sequencecomprises DNA.

The invention also includes cloning and expression vectors containingthe DNA sequences of the invention. Such expression vectors willincorporate the appropriate transcriptional and translational controlsequences, for example enhancer elements, promoter-operator regions,termination stop sequences, mRNA stability sequences, start and stopcodons or ribosomal binding sites, linked in frame with the nucleic acidmolecules of the invention.

Additionally, in the absence of a naturally-effective signal peptide inthe protein sequence, it may be convenient to cause the recombinantprotein to be secreted from certain hosts. Accordingly, furthercomponents of such vectors may include nucleic acid sequences encodingsecretion signalling and processing sequences.

Vectors according to the invention include plasmids and viruses(including both bacteriophage and eukaryotic viruses). Many such vectorsand expression systems are well known and documented in the art.Particularly suitable viral vectors include baculovirus-, adenovirus-and vaccinia virus-based vectors.

The expression of heterologous polypeptides and polypeptide fragments inprokaryotic cells such as E. coli is well established in the art; seefor example Molecular Cloning: a Laboratory Manual: 2nd edition,Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or DNAcloning: a practical approach, Volume II: Expression systems, edited byD. M. Glover (IRL Press, 1995). Expression in eukaryotic cells inculture is also an option available to those skilled in the art for theproduction of a heterologous proteins; see for example O'Reilly et al.,(1994) Baculovirus expression vectors—a laboratory manual (OxfordUniversity Press) or DNA cloning: a practical approach, Volume IV:Mammalian systems, edited by D. M. Glover (IRL Press, 1995).

Suitable vectors can be chosen or constructed for expression of peptidesor proteins suitable for use in accordance with the present invention,containing the appropriate regulatory sequences, including promotersequences, terminator sequences, polyadenylation sequences, enhancersequences, marker genes and other sequences as appropriate. Vectors maybe plasmids, viral e.g. bacteriophage, or phagemid, as appropriate. Forfurther details see Molecular Cloning: a Laboratory Manual. Many knowntechniques and protocols for manipulation of nucleic acid, for example,in the preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al, eds., (John Wiley & Sons, 1992)or Protein Engineering: A practical approach (edited by A. R. Rees etal., IRL Press 1993). For example, in eukaryotic cells, the vectors ofchoice are virus-based.

A further aspect of the present invention provides a host cellcontaining an antibody or equivalent ligand according to the firstaspect of the invention or encoding a peptide, oligopeptide, polypeptideor protein according to the second aspect of the invention. A stillfurther aspect provides a method comprising introducing such nucleicacid into a host cell or organism. Introduction of nucleic acid mayemploy any available technique. In eukaryotic cells, suitable techniquesmay include calcium phosphate transfection, DEAE-Dextran,electroporation, liposome-mediated transfection or transduction usingretrovirus or other viruses, such as vaccinia or, for insect cells,baculovirus. In bacterial cells, suitable techniques may include calciumchloride transformation, electroporation or transfection usingbacteriophage.

Introduction of the nucleic acid may be followed by causing or allowingexpression from the nucleic acid, e.g. by culturing host cells underconditions for expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

Transgenic animals transformed so as to express or overexpress in thegerm line one or more compounds as described herein form a still furtheraspect of the invention, along with methods for their production. Manytechniques now exist to introduce transgenes into the embryo or germline of an organism, such as for example, illustrated in Watson el al.,(1994) Recombinant DNA (2nd edition), Scientific American Books.

Therapeutic Implications of the Invention and Application to UnansweredQuestions in Diabetes

The following are given by way of example and not by way of limitation.

Impaired Glucose Counterregulation

A major problem in the management of IDDM patients is the occurrence ofhypoglycaemia which may be partially iatrogenic due to intensive insulintreatment leading to hypoglycaemia unawareness, but is mainly due tocompromised glucose counterregulation. Defective glucosecounterregualtion is the result of the combined deficiencies of theglucagon and epinephrine responses to falling glucose levels. It hasbeen demonstrated that scrupulous avoidance of hypoglycaemia can reversehypoglycaemia unawareness but not defective counterregulation (Cryer,1995).

Not only longstanding but also newly diagnosed IDDM patients haveimpaired counterregulation. A comparison of counterregulatory responsesin twenty children with new onset IDDM (5-6 days) and 47 children withlong standing IDDM, revealed that glucagon responses to hypoglycaemia inboth groups were lower than in control subjects. Epinephrine responseswere also reduced in new IDDM patients compared to controls (Hoffman elal., 1994).

It is proposed herein that the reason for these counterregulatorydefects is the persistence of auto-anti-anti-Vβ antibodies in IDDMpatients causing downregulation of the signalling molecules describedabove and abrogating the response of alpha cells and adrenal medullarycells to falling glucose levels. This is in keeping with the observationof loss of anti-anti-Vβ staining of pancreatic sections from a newlydiagnosed diabetic patient who died accidentally (see experimentalsection p39). It is also analogous to the findings of Brett el al.(1996) that treatment of rheumatoid arthritis patients with Campath-1Hwhich is against the GPI-linked CD52 protein, resulted in disappearanceof CD52 and other GPI-linked proteins on both T cells and B cells ofsome of the patients treated. The CD52-negative B cells disappeared fromthe circulation within 3 months; however, the CD52-negative T cellspersisted for at least 20 months. Therefore preventing the perpetuationof these antibodies in IDDM patients should ameliorate counterregulatorydefects preventing hypoglycaemia. Blocking their development from birthshould prevent IDDM in susceptible individuals, NIDDM should be curedentirely. This will be accomplished by a method analogous to theadministration of anti-D immunoglobulin (anti-D Ig) to Rh-negativemothers carrying Rh-positive foetuses (Davey, 1979). Possible mechanismswhich are involved in the blocking of antibody production in this typeof therapy are discussed by Heyman (1990). As a further measure,immunising individuals with the pathogenic antibodies should generateprotective anti-idiotypic antibodies which can then complex with thepathogenic antibodies when they arise.

Diabetic Nephropathy

Renal involvement in type I diabetes is characterised by epithelial andbasement membrane hypertrophy of the glomeruli and tubules andaccumulation of extracellular matrix components in the glomerularmesangium (Lane el al., 1990). Progression of the disease leads toobliteration of the glomerular capillary lumen, proteinuria and loss offiltration. Hyperglycaemia and production of TGF-β (transforming growthfactor) have been implicated in diabetic nephropathy. High glucoseconcentrations increase TGF-β mRNA and protein in cultures of mesangialand proximal tubular cells; the TGF-β indirectly mediates the effects ofglucose on cell growth and collagen synthesis. Administration ofantiserum against TGF-β has been shown to suppress experimentalglomerulonephritis (Border, 1990).

It is likely that hyperglycaemia induces TGF-β expression early indiabetes; this is supported by the fact that both in human diabetes andin the BB and NOD models, increased renal expression of TGF-β has beendemonstrated within a few days after the onset of hyperglycaemia andrenal hypertrophy (Yamamoto el al., 1993; Sharma and Ziyadeh, 1994).

The binding of TGF-β to its receptor is assisted by a membrane anchoredproteoglycan (β glycan) that presents TGF-β to the type II signallingreceptor, a transmembrane serine/threonine kinase (Lopez-Castillas etal., 1994). Betaglycan has an extracellular region which is shed bycells and can bind TGF-β but cannot present it to the signallingreceptor and consequently acts as a potent inhibitor of its action.Betaglycan belongs to a family of proteins which includes uromodulin andthe pancreatic secretory granule membrane GP-2. The role of theuromodulin related region in TGF-β binding has been demonstrated(Fukushima et al., 1993).

The α cell molecule related to GP-2 (product of clone 5.3) which existsin soluble and membrane bound form may be one of the proteins involvedin the inhibition of TGF-β action. The down regulation of thesemolecules due to prolonged action of the pathogenic antibodies mayresult in the abrogation of TGF-β inhibition, thus promoting its trophicproperties in the kidney. Administration of soluble peptides of themolecules recognised by the pathogenic antibodies would have the dualrole of inhibiting TGF-β and suppressing antibody production.

Pancreas Transplantation

Transplantation is increasingly being performed to treat type 1diabetics prone to severe hypoglycaemic episodes. This has the dual roleof establishing insulin independence and partially restoringnormoglycaemia. If this procedure, however, is carried out withoutcounteracting the underlying diabetogenic conditions, the glucosecounterregulatory problems will reemerge with each successive episode ofpathogenic antibody development.

In a recent study of 13 successful pancreas transplant patients using astepped hypoglycaemic clamp technique, it was demonstrated that glucagonresponses to hypoglycaemia were restored. However, both fasting andstimulated glucagon levels were significantly greater in the pancreastransplant recipients compared to normal controls or kidney transplantrecipients. Furthermore, C-peptide levels were also raised compared toall other groups (Kendall et al., 1997). The authors did not comment onthese observations which are reminiscent of a prediabetic condition.They reported, however that epinephrine responses to hypoglycaemia wereimproved in the pancreas transplant recipients but were significantlylower than in healthy control subjects or non-diabetic kidney transplantrecipients. It is clear from these observations that transplantation ofa healthy pancreas into a diabetic patient sets the clock back to theprediabetic state in terms of both the α cells and the adrenal response.A potential diabetic pancreas transplant patient must be treated toprevent further episodes of rises in pathogenic antibody titres prior totransplantation to ensure complete success of the procedure.

Autonomic Neuropathy

Diabetes of long standing may be complicated by autonomic neuropathywhich is irreversible and distinct from hypoglycaemia unawareness(Cryer, 1994; Dagogo-Jack et al., 1993). The elimination ofhypoglycaemia by means of pancreas transplantation in the study ofKendall et al., (1997) improved both the epinephrine response andhypoglycaemic symptom recognition despite the persistence of cardiacautonomic dysfunction. A norepinephrine response, however, which wasabsent in the long-standing diabetic patients was not restored bypancreas transplantation. Although the reactivity of the diabetogenicmonoclonal antibodies against the neuronal cell bodies in the autonomicganglia has not yet been tested, it is anticipated that the antigensthey recognise will also be present on these cell bodies. The expressionof unique sets of GPI-linked proteins on different primary neurones hasbeen demonstrated. Some of these have been shown to correspond todifferent ensheathment characteristics (Rosen et al., 1992). Suchmolecules will have, similar signalling properties and may be affectedsimilarly to those on α cells and adrenal medullary cells.

The GPI-linked membrane protein, ciliary neurotrophic factorreceptor(CNTF) has already been implicated in some forms of peripheraldiabetic neuropathy. In hyperglycaemia induced by galactose feeding orstreptozotocin treatment of experimental animals, the levels ofCNTF-like activity in sciatic nerve were reduced after 1-2 months ofhyperglycaemia. This has been associated with reduction of CNTF proteinbut not mRNA. Deficits of CNTF resulting from Schwann cell injury maycontribute to certain functional and structural abnormalities inexperimental diabetic neuropathy. Some of these abnormalities are due toaldose reductase (AR) metabolism of hexoses and can be prevented by ARinhibitors. However, CNTF deficiency was only partially restored bythese inhibitors indicating that factors other than polyol accumulationdue to AR activity are involved in reduction of CNTF expression (Mizisinet al., 1997). This demonstrates that GPI-linked molecules may play asignificant role in peripheral diabetic neuropathy as well as autonomicneuropathy.

Therapeutic Implications of the Invention and Application to UnansweredQuestions in SLE and the Primary Anti-phospholipid Syndrome

The following are given by way of example and not by way of limitation.

Antibodies with specificity against anionic phospholipids such ascardiolipin have been associated with thrombosis, recurrent foetal lossand thrombocytopenia (Harris el al., 1983; Cowchock el al., 1986; Harriset al., 1986). Similar claims have been made for systemic lupuserythematosus (SLE) associated antibodies called lupus anticoagulantwhich are detected by their partial thromboplastin time (Thiagarajan etal., 1980; Love and Santoro, 1990). The anti-coagulant effect has beenshown to be due to a specific reactivity of these antibodies withanionic phospholipids (Sammaritano et al., 1990). In addition, SLEpatients have antibodies against native double-stranded DNA (dsDNA)which serve as diagnostic markers for SLE (Veinstein et al., 1983). Mostpatients with anti-phospholipid antibodies have SLE or a relatedautoimmune condition; some, however, have no other detectable diseaseand are considered as having a ‘primary anti-phospholipid syndrome’(PAPS) (Asherson el al., 1989; Branch et al., 1990). In recent years thepathogenic significance of these antibodies has been established byinducing foetal loss in pregnant mice by passive transfer of humanpolyclonal antiphospholipid antibodies (Branch el al., 1990). PAPS hasalso been induced in naive mice by passive transfer of human polyclonaland mouse monoclonal anti-cardiolipin antibodies (Blank et al., 1991).

Anti-phospholipid or anticardiolipin antibodies also occur in a numberof neurological conditions and their role has been emphasised in focalcerebral ischaemia, migraine, chorea, seizures and other conditions(Levine and Welch, 1987).

To date, the origin of anti-phospholipid or anti-dsDNA antibodiesremains unknown. Studies in this regard appear to focus on the ligandbinding properties of anti-phospholipid antibodies. Polyclonalanti-phospholipid antibodies from patients cross-react with the majorityof anionic phospholipids (Lafer et al., 1981; Pengo et al., 1987).Attention however, was diverted to other ligands when monoclonalantibodies which bind to polynucleotides such as DNA were shown to bindalso to cardiolipin and other anionic phospholipids (Schoenfeld et al.,1983; Rauch et al., 1984; Smeenk et al., 1987). This cross-reactivity isthought to be due to similarity in chemical structure of DNA andphospholipids which both contain phosphodiester-linked phosphate groupsthat are separated by three carbon atoms (Lafer et al., 1981).Lipoteichoic acids from gram-positive bacteria and endotoxin fromgram-negative bacteria also contain phosphate esters and such moleculesin foreign antigens are considered to be possible triggers for thegeneration of anti-phospholipid antibodies (Carroll et al., 1985).

Recently, the development of anti-dsDNA antibodies has been shown tocorrelate with frequent polyoma virus reactivations in some SLEpatients. However, high titres of anti-dsDNA were also detected in theabsence of viral DNA (Rekvig et al., 1997).

It has already been demonstrated herein that both cardiolipin andanti-dsDNA reactivities are encompassed within the binding specificitiesof anti-anti-TCR Vβ antibodies (see Table 3. For methods seeexperimental section). This is a characteristic of both the polyclonalantibodies from mice immunised with anti-TCR Vβ monoclonal antibodiesand the anti-anti-TCR Vβ monoclonal reagents produced from suchimmunised mice. Furthermore, the said polyclonal mouse antisera had apowerful anticoagulant effect.

The potential mechanisms for the pathophysiological development ofanti-anti-TCR Vβ antibodies have already been discussed (see page 12).The use of polyclonal or monoclonal anti-anti-TCR Vβ antibodies inpreventing their development or the induction of protective antibodieshas been pointed out (see page 25). Such methods should prevent thedevelopment of the combination of pathogenic anti-DNA andanti-phospholipid antibodies resulting in the alleviation of thediseases caused by these antibodies.

Application of the Invention to Further Diseases of HormonalDysregulation and Conditions Where β Cell Dysfunction orHyperinsulinaemia and Insulin Resistance are Present

As indicated previously the anti-anti-Vβ antibodies bind to islet αcells and other endocrine organs suggesting that its target moleculesare involved in their secretory mechanisms. This would explain thefinding that autoimmune endocrine disease can effect more than one organin a single patient or autoantibodies against a clinically ‘unaffected’organ can be present. The diseases which can coexist are hypothyroidism,hyperthyroidism (Grave's disease), diabetes mellitus, Addison's disease,primary hypogonadism, autoimmune gastritis and pernicious anaemia amongothers, the disease profile presumably reflecting the individual'sgenetic susceptibility.

The following are given by way of example and not limitation

Autoimmune Thyroid Disease

The incidence of autoimmune thyroid disease is substantially higheramong patients with IDDM than in the general population (Payami andThomson, 1989). Abnormal glucose tolerance and increased hepatic glucoseproduction is often observed in hyperthyroidism (Wennlund et al. 1986).The accelerated gluconeogenesis is indicative of hyperglucagonaemiawhich was reported both in the basal state and after insulin infusion in8 newly diagnosed hyperthyroid subjects by Moghetti et al. (1994). Also,the percentage decrease in glucagon levels after glucose administrationor a meal is significantly less among hyperthyroid patients whether theyare hyperglycaemic or not (Kabadi and Eisenstein, 1980; Bech et al.1996). Insulin secretion is also dysregulated in hyperthyroidindividuals. In a variety of conditions such as during a hyperglycaemicclamp (O'Meara el al. 1993), in the fasting state and after a meal (Bechet al. 1996) immunoreactive insulin concentrations were higher inthyrotoxic patients compared to controls. The rise in immunoreactiveinsulin was accounted for by increased proinsulin secretion. There isalso evidence of increased secretion of ACTH (adrenocorticotrophichormone), cortisol and growth hormone in hyperthyroidism (Moghetti etal. 1994; Gallagher et al. 1971) which is consistent with the hypothesisof dysregulation of the normal negative feedback control of hormonesecretion due to the binding of anti-anti Vβ antibodies to targetantigens on these organs. The dysregulated glucagon and insulinsecretion in thyrotoxicosis is similar to the prediabetic and diabeticstates; in analogous fashion the nocturnal TSH surge is blunted in mostdiabetic patients (Coiro et al. 1997).

Polycystic Ovary Syndrome (PCOS)

There is persistent enhanced early insulin response to intravenousglucose in women with PCOS which indicates a primary abnormality ofinsulin secretion (Holte, 1995). Such women also have a hyperglycaemicand hyperinsulinaemic response during an oral glucose tolerance test(OGTT) (Dunaif et al. 1987). Golland et al. (1990), however, reportedthat PCOS women had blunted glucagon responses in spite ofhyperglycaemia during OGTT. This indicates that a second line glucosecounterregulatory hormone i.e. epinephrine must be increased in PCOSwomen. Consistent with this is the adrenal hyperandrogenism found inhalf of the women with androgen excess (Ehrmann et al. 1992). The effectof adrenaline on steroidogenesis has been demonstrated both in perfusedislolated adrenals and at the molecular level (Ehrhart-Bornstein et al.1994; Guse-Behling et al. 1992). The histological demonstration byimmunostaining of the intermingling of adrenal cortical cells within theentire adrenal medulla and vice versa confirms the role of the adrenalmedulla as a regulator of adrenocortical function by a paracrinemechanism (Bornstein el al. 1994). The molecules recognised by thepathogenic autoantibodies described in this invention are abundantlyrepresented on the adrenal medullary cells (FIG. 3). Such autoantibodiescan be the cause of increased adrenaline secretion causing the adrenalhyperandrogenism in PCOS.

Adrenal hyperandrogenism frequently coincides with ovarianhyperandrogenism which is generally accompanied by LH augmentation. Theabnormal pattern of ovarian steroidogenesis can only partly be explainedby LH hyperstimulation of thecal cells and a hyperresponse to GnRH.Insulin and insulin like growth factors augment the androgenic responseof thecal cells to LH by increasing levels of the rate determiningenzymes in ovarian steroidogenesis and reversing LH induceddownregulation of these enzymes (Hernandez et al. 1988; Magoffin et al.1990). Therefore, hyperinsulinaemia has been proposed as the majorcandidate of ovarian dysregulation (Ehrmann et al. 1995).

Hyperinsulinaemia also appears to have a role in adrenalhyperandrogenism, however not directly but by synergising with ACTHstimulation (Moghetti et al. 1996). The secretion of pituitaryglycoprotein hormones is pulsatile and the disruption of their pulsescan alter reproductive function (Samuels et al. 1990; Santoro et al.1986). It has been demonstrated that cultured pituitary lactotrophsexpress GPI-linked molecules which are rapidly hydrolysed by treatmentwith TRH (Benitez et al. 1995). Phospholipase C inhibition prevents theaction of TRH and second messenger generation (Perez et al. 1997). Therelease of ACTH from rat anterior pituitary cells was shown to beprevented by inhibiting phospholipase C activity (Won and Orth, 1995).The effects of ACTH are also mimicked by phospholipase C (Foster andVeitl, 1995). Villa et al. (1995) reported that the accumulation ofaldosterone in adrenocortical cells was inhibited in a dose-dependentmanner by insoitol phosphoglycans demonstrating the regulatory role ofthese molecules. These observations demonstrate the far reaching effectsof blocking sites of phospholipase C action by pathogenic anti-anti-Vβautoantibodies described herein.

Obesity

Hyperinsulinaemia is characteristic of both juvenile and adult obesity.Le Stunff and Bougneres (1994) reported a 76% increase in insulinresponse to a standard meal in children with long or short duration ofobesity; fasting insulin levels increased with duration of obesity.Obese children of long or short duration are also hyperglycaemic after astandard meal test compared to controls which is consistent with areport of increased gluconeogenesis in recently obese children (LeStunff and Bougneres 1996).

Increased postprandial insulin increment has been shown to persist inwomen with massive obesity after normal body weight was achieved(Fletcher, et al. 1989). Therefore, hyperinsulinaemia appears to be aprimary abnormality leading to obesity. In adult obesity,hyperinsulinaemia is also associated with increased levels of free fattyacids both during fasting and postprandial states (Golay et al. 1986).Increased gluconeogenesis and hyperinsulinaemia are likely to be theresult of dysregulated glucagon secretion in obesity. Borghi et al.(1984) reported that glucose failed to suppress glucagon secretion inobese subjects. Golland el al. (1990) demonstrated that obese women hada significantly greater glucagon response at 60, 90 and 120 minutesafter oral glucose loading than did non obese subjects. Both theseobservations demonstrate the lack of regulatory signals in pancreatic αcells analogous to prediabetic and diabetic conditions.

Cushing's Syndrome

This disease is commonly associated with glucose intolerance, diabetes,central obesity. hirsutism and elevated arterial blood pressure. Themain diagnostic feature is hypercortisolism which may result from longstanding ACTH hypersecretion in 20-40% of patients (Doppman el al.1988); this can occur in the absence of a pituitary adenoma andincreased cortisol secretion can be due to unilateral or bilateraladrenal hyperplasia with or without autonomously secreting micro ormacro nodules (Hermus el al. 1988).

In a recent cross-sectional study of 90 patients with obesity anddiabetes, the prevalence of Cushing's syndrome was reported to be 3.3%(Leibowitz et al. 1996). Pre-clinical and sub-clinical cases ofCushing's which present as poorly controlled diabetes add to this figureconsiderably. In analogous fashion mild chronic hypercortisolism hasbeen reported in type 1 diabetes reflected by elevated fasting cortisoland urinary free cortisol and an increased response to ovinecorticotropin-releasing hormone (Roy el al. 1993).

ACTH hypersecretion can occur in the absence of a pituitary adenoma butin the presence of hypercortisolaemia (Grant and Liddle, 1960)suggesting a dysregulation of the normal negative feedback controlSeveral reports are indicative of the role of GPI-linked molecules andinositol phosphoglycans released by the activation of phospholipase C inthe regulation of both pituitary hormone secretion and the secretion ofthe hormones that they stimulate from the adrenals, thyroid, gonads etc.(Fanjul el al. 1993; Shaver et al 1993; Villa et al. 1995;). It istherefore anticipated that the autoantibodies described herein will havepathogenic effects ranging from disruption of pulsatile secretion ofhormones to inhibited or exaggerated secretion and even the formation oftumours as antibodies to GPI-linked molecules have also been shown toinduce cell proliferation by causing loss of inhibitory input toactivating signals (Robinson and Hederer, 1994; Benitez el al. 1995).

Metabolic Syndrome X and Cardiovascular Diseases

Syndrome X is the combination of hyperinsulinaemia, glucose intolerance,increased very low density lipoproteins (VLDL) and triglyceridesdecreased high density lipoproteins (HDL) and hypertension. Centralobesity is also associated with this syndrome. The primary causalabnormality of this syndrome is considered to be insulin resistance(Reaven, 1988; Reaven, 1995). Hrnciar et al. (1992) estimated thepresence of the Syndrome in 5-10% of the general population, in 15-30%of patients with arterial hypertension, in 65-90% of NIDDM, in 10-20% ofhirsutic women and in 30-50% of patients with myocardial infarction.Piedrola et al. (1996) reported that 82.5% of 40 newly diagnosedcoronary artery disease patients were insulin resistant and 27 of the 40had an abnormal OGTT. Hyperinsulinaemia and insulin resistance correlatewith the severity of peripheral vascular, coronary and carotid arterydisease (Standl 1995; Reaven 1995) and are also involved inmicrovascular angina and exercise induced coronary ischaemia(Vertergaard et al. 1995).

In a survey of cardiovascular disease and Syndrome X in 2930 subjectsFerrannini et al. (1991) reported that isolated forms of each conditionof the syndrome were rare but were always associated withhyperinsulinaemia suggesting that this is the key feature of thesyndrome. Sowers et al., (1993) have also suggested thathyperinsulinaemia may contribute to the development of hypertension bypromoting atherosclerosis and vascular remodelling. Insulin resistancehas been observed to be associated with increased carotid wall thickness(Suzuki et al. 1996) and carotid artery plaques (Laakso et al. 1991). Arecent prospective population-based study by Salonen et al. (1998)supports the hypothesis implicating insulin resistance in the etiologyof hypertension and dyslipidemia. Moller et al. (1996) demonstrated thata pure defect in muscle insulin receptor-mediated signalling causedinsulin resistance, hyperinsulinaemia, obesity, increased plasmatriglycerides and free fatty acids in transgenic mice. In NIDDM musclebiopsies indicate a generalised deficiency of inositol phosphoglycanmediators of insulin action (Asplin et al. 1993). The pathogenicantibodies described herein could cross-react with such mediators andinduce insulin resistance both by downregulating them and also bydisrupting pulsatile secretion of insulin.

Immunologically Mediated Multisystem Diseases

Hyperinsulinaemia and insulin resistance have also been been shown to beprominent in multisystem diseases such as systemic lupus erythematosusand progressive systemic sclerosis. The serum fasting insulin levels in21 such patients were double that of normal controls and they hadsignificantly higher triglycerides and lower HDL cholesterol levels(Mateucci et al. 1996)

Cancer

Hyperinsulinaemia, a diabetic pattern of glucose tolerance, an increasedrate of HGP and insulin resistance are associated with many cancersincluding breast, colorectal, gastrointestinal, sarcoma, endometrial,prostate, head neck and lung (Tayek 1992; Copeland, Leinster et al 1987;Copeland, Al-Sumidaie et al. 1987; Tayek 1995; Nagamani et al. 1988).Bruning et al. (1992) demonstrated that the log relative risk of breastcancer was linearly related to the log C-peptide levels. This wasindependent of BMI (body mass index) or WHR (waist to hip ratio); the223 women with stage I or stage II breast cancer were insulin resistantand had significantly higher C-peptide levels than the 441 controls. Ina recent study of 2569 histologically confirmed cases of breast cancerand 2588 control women an association of breast cancer with late onsetdiabetes has also been noted (Talamani et al. 1997). The direct role ofinsulin in promoting tumours has been shown in a rat model of colontumours (Tran et al. 1996).

Cancer cachexia also appears to be characterised by glucose intolerance,postabsorptive hyperglycaemia, reduced total body glucose utilisationconsistent with insulin resistance and augmented peripheral lactateproduction. The insulin to glucagon ratio is also reduced; increasedcirculating glucagon levels are associated with the tumour-bearing state(Cersosimo el al. 1991) which is consistent with the increased HGP inmany cancers. Bartlett et al. (1995) demonstrated that increasing theinsulin/glucagon ratio by hormone therapy selectively supported hostanabolism and inhibited tumour growth kinetics in a rat model.Therefore, preventing the development of the diabetogenic complex ofmetabolic derangements will reduce the incidence of cancers andalleviate symptoms of cancer cachexia.

Diagnostic, Prophylactic and Therapeutic Uses of the Invention

The following are given by way of illustration and not limitation.

The invention will be applied to the prevention and treatment ofautoimmune and related diseases by injecting pharmaceutical preparationsof the monclonal or polyclonal anti-anti-Vβ antibodies or equivalentligands, the peptide fragments or molecules recognised by theseantibodies and functionally active vectors containing RNA or DNAsequences coding for such peptides or molecules.

Injection of antibody will be designed to prevent the development ofautoantibodies of the same specificity by feedback mechanismssuppressing existing B cells or by an idiotypic network of antibodydevelopment giving rise to protective antibodies (see pages 17 and 25).Soluble peptides or other target molecules recognised by the pathogenicanti-anti-Vβ antibodies will also be used to induce low dose tolerance,the specific blocking of already activated B cells (see page 17) or inlarger predetermined doses to block the action of the mediators ofspecific nephropathy such as TGF-β (see page 27). Vectors containingappropriate nucleic acid sequences will also be injected bypredetermined regimens to allow the long term in vivo secretion ofsoluble products which will function as tolerogens. The peptides,proteins or other molecules recognised by the anti-anti-Vβ pathogenicantibodies and the anti-Vβ immunogens used in the generation of theseantibodies will be used in the development of diagnostic kits to detectthe presence of auto-anti-anti-Vβ antibodies in blood, plasma, serum,saliva or other body fluids to ascertain susceptibility to autoimmunedisease or as prognostic indicators of the progression of disease ortreatment efficacy.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Staining of a normal human pancreatic section with a monoclonalanti-anti-Vβ antibody detected by a fluoresceinated second reagent.

FIG. 2. Staining of a normal human thyroid section with a monoclonalanti-anti-Vβ antibody detected by a fluoresceinated second reagent.

FIG. 3. Staining of a normal human adrenal section with a monoclonalanti-anti-Vβ antibody detected by a fluoresceinated second reagent.

FIG. 4. Staining of a normal human intestine section with a monoclonalanti-anti-Vβ antibody detected by a fluoresceinated second reagent.

FIG. 5. Staining with an anti-anti-Vβ monoclonal antibody andfluoresceinated second reagent of a pancreas section from a child whodied at diagnosis of diabetes from uncontrolled ketoacidosis.

FIG. 6. Sequence of the ESRP1 gene (SEQ ID NO:1).

FIG. 7. Predicted protein sequence for the ESRP1 proteins (SEQ IDNOS:2-4).

EXPERIMENTAL

The following examples are given by way of illustration not by way oflimitation.

Development of Monoclonal Antibodies

Mice were immunised intraperitoneally (ip) with 4 weekly injections of0.1 ml monoclonal antibody hybridoma culture supernatant against TCR Vβspecificities. The spleens were then removed and single cell suspensionswere prepared. The cells were fused with Sp2 myeloma cells usingstandard techniques known to workers in the field and related fields.The antibody producing clones were identified in ELISA using peroxidaseconjugated anti-Ig reagents. The clones were further screened againstthe immunising reagent, double and single stranded DNA and anionicphospholipids. Methods used were standard techniques known to workers inthe field.

Detection of Anti-phospholipid Antibodies

Flexible 96 well flat bottom plates (Falcon, Becton-Dickinson) werecoated with 50 μl of 50 μg/ml in ethanol of cardiolipin,phosphatidylcholine, phosphatidylserine and 50 μg/ml in methanol ofphosphatidylinositol (Sigma). Control wells were coated with diluentalone. The plates were left at 4° C. until evaporation. Unbound siteswere blocked with 0.1% human serum albumin (HSA) in phosphate bufferedsaline(PBS). The plates were washed with PBS containing 0.05% Tween 20(RTM) and incubated with serial dilutions of sera in PBS Tween (RTM) orMoAb culture supernatants. After incubation for 1 hour at 37° C. orovernight at 4° C., the plates were washed again as above and the boundantibodies were detected using a 1:500 dilution of biotinylatedanti-mouse Ig (Amersham International plc), incubated for 30 minutes at37° C. followed after appropriate washing by a further 30 minutesincubation at 37° C. with 1:500 streptavidin-biotinylated horseradishperoxidase complex (Amersham International plc). O-phenylenediamine(Sigma) was used as substrate and the colour was read at 450 nm using anAnthos ELISA reader.

Detection of Anti-DNA Antibodies

Wells of 96 well flat bottom flexible plates were coated first with 50μg/ml poly-1-lysine in water by incubating for 1 hour at roomtemperature. After discarding the poly-1-lysine solution, 50 μl ofsingle stranded or double stranded DNA (Sigma) solution 10 μg/ml in PBScontaining 1 mM EDTA was added to each well and the plates incubated for1 hour at room temperature. The plates were washed in PBS. Remainingbinding sites were blocked with 0.1% HSA in PBS. The plates were washedwith PBS containing 0.05% Tween 20 (RTM) and incubated with serialdilutions of sera or MoAb culture supernatants. Binding of antibody wasdetected as described above for anti-phospholipid antibodies.

Testing for the Anticoagulant Effect of Anti-anti-Vβ Antibodies

Antisera from several strains of inbred mice immunised by variousanti-TCR Vβ monoclonal antibodies were tested. Five μl of each antiserumwas mixed with 95 μl hard spun normal human plasma and incubated at 37°C. for 1 hour. To this was added 100 μl of appropriately dilutedRussell's viper venom (Diagen) and 100 μl of diluted platelet substitute(Diagen) and incubated for 30 seconds. 100 μl of 0.025M CaCl₂ was thenadded and the clotting time measured. Clotting time in the presence ofnormal mouse serum was approximately 55 seconds while in the presence ofthe said immune sera the clotting time ranged from 10 to 30 minutes. Acontrol murine antiserum did not prolong the clotting time above that ofnormal mouse serum.

Staining of Diabetic Pancreas Sections

Pancreas sections from a recently diagnosed insulin dependent diabeticpatient who died accidentally (from diabetes unrelated causes) andpancreas sections from normal cadaveric organ donors were stained withanti-anti-Vβ monoclonal antibodies. The normal pancreas sections showedintraislet staining as expected (see FIG. 1) but the diabetic pancreaseither did not stain at all or stained very faintly. This indicates thatin this patient the relevant α cell antigens were downregulated orswitched off.

In contrast to this, anti-anti-Vβ staining of pancreatic sections fromthree children who died at diagnosis from keto-acidosis showed aproliferation of positive staining cells outside the confines of islets(FIG. 5). This may have been due to large amounts of autoantibodyreacting in vivo with the laminin binding-like protein or other targetproteins causing proliferation and migration of the α cells outside ofthe islets.

Both the scenarios described above could fit into a spectrum ofresponses by individuals of different genetic constitution to causefulminant uncontrolled keto-acidosis and death or IDDM in individualswith fragile β cells and NIDDM in those with more robust β cells.

Effect of Monoclonal Anti-anti-V Beta Antibodies on Intact Human Isletsin Vitro

Separated human islets from a cadaveric organ donor were washed in RPMI1640 medium containing 10% foetal calf serum and cultured at aconcentration of approximately 200 islets per well in the same medium in24-well plates. Three days later the medium from duplicate wells wascarefully removed and stored at −20° C. The control wells were thencultured with medium alone and the test with hybridoma culturesupernatant containing anti-anti-Vβ diluted with an equal volume offresh medium. After 24 hrs the supernatant in each well was removed andstored as above and replenished with medium alone or hybridomasupernatant diluted as above. This was repeated daily for 2 weeks exceptthat the supernatants were not removed during the week-end. At the endof the experimental period, the insulin in the stored samples wasmeasured using a DAKO insulin kit according to the manufacturer'sinstructions. The results demonstrated that the insulin levels in thetest and control wells were almost identical at the start of theexperiment. Twenty four hours after the addition of antibody the insulinlevel in the test well rose considerably higher than in the controlwell. On the third day insulin secretion in the antibody containing welldropped to approximately 50% of the control. On the fourth day insulinsecretion in the test well was again above the control well, while onthe fifth day the levels were similar. The results given in opticaldensity (OD) readings in Table 1 demonstrate that while insulin releasein the control well was fairly constant, there were sharp fluctuationsin the test well during the first experimental week. During the secondweek insulin in the test well dropped and by the tenth day no secretioncould be detected while in the control well secretion was well above thebackground OD reading. Even though further measurements of secretion inthe control wells were not carried out, the slow rate of decline ofinsulin secretion in the control well indicates that secretion couldhave carried on for several more days.

TABLE 1 Effect of monoclonal anti-anti-TCR Vβ antibodies on human isletsin vitro Optical Density Day No. Test Control 1 2.117, 2.063 2.042,1.848 2 2.784, 2.751 2.143, 2.044 3 1.236, 1.057 2.256, 2.240 4 2.513.2,377 2.124, 2.187 5 2.446, 2.450 2.506, 2.545 8 0.699 1.114 9 0.7771.049 10 0.585 0.979 11 0.482 0.842

Optical density was measured using an Anthos 2001 plate reader at 450 nmwith reference filter at 650 nm. Optical density for culture medium was0.532.

Demonstration of Naturally Occurring Anti-anti-Vβ Autoantibodies inHuman Sera

Anti-anti-Vβ antibodies were generated from spleens of mice immunisedwith culture supernatant from hybridoma cell lines secreting anti-TCR Vβantibodies. These anti-anti-Vβ monoclonals were shown to bind to theanti-Vβ immunogen in ELISA; therefore the use of this immunising reagentas antigen to detect the presence of auto anti-anti-Vβ antibodies inhuman sera was examined. The anti-Vβ immunogen was used to coat 96-wellflat-bottomed plates overnight, the unbound sites blocked and human seraadded in 1/30 dilution to the wells. After 2 hrs, incubation the plateswere washed and the binding of the human serum detected using aperoxidase conjugated anti-human Ig.

Table 2 depicts results with sera obtained from three prediabetic donorswho subsequently became diabetic. The serum samples from donor 3 werefortuitously spaced and demonstrate the highest level of autoantibody ayear before diagnosis. A rise in the index of binding (Test OD/ControlOD) from 4.4 to 6.1 occurred within 7 months of the first sample anddropped to 2.9, 2 months before diagnosis. This demonstrates thetransient nature of this autoantibody and that it may not be long-termpersistence that leads to disease development but perhaps severalepisodes of rises in titre due to viral or other infections leading toT-cell proliferation and the appearance of abnormal GPI-linked TCR Vβchains. The autoantibodies appear to have persisted, however, for atleast seven months to a year at high levels in patient 3. This may haveled to downregulation of the signalling molecules on the pancreatic,cells as mentioned earlier (pages 25 and 40).

TABLE 2 Anti-anti-TCR Vβ autoantibodies are present in human seraDiagnosis Optical Density Donor Serum of IDDM Test/ No. date date Testantigen Control Control(Medium) 1  5/1989  1/1991 0.082, 0.080 0.124,0.134 2  2/1989  1/1993 0.087, 0.076 1.5 0.058, 0.054  6/1989 0.079,0.074 0.076, 0.063 3  5/1987 12/1988 0.074, 0.072 4.4 0.016, 0.01712/1987 0.109, 0.097 6.1 0.016, 0.018 10/1988 0.060, 0.057 2.9 0.021,0.019

The test antigen was culture supernatant from an anti-TCR Vβ producingmonoclonal cell line.

The Test/Control index was obtained by dividing mean OD test by mean ODcontrol.

TABLE 3 Binding specificities of monoclonal anti-anti-TCR Vβ antibodiesOptical Density Well coat reagent Experiment 1 Experiment 2 Anti-TCR Vβ(Immunogen) 0.413 0.399 Culture medium (Control) 0.046 0.040 Cardiolipinin ethanol 1.002 0.998 Ethanol (Control) 0.156 0.126 dsDNA 0.210 0.242Poly-l-lysine (Control) 0.119 0.129

Optical density was measured using an Anthos 2001 plate reader at 450 nmwith reference filter at 650 nm. The anti-TCR Vβ was used as culturesupernatant.

Screening of Pancreas Library with Monoclonal Antibodies (MoAbs)

Libraries in λgt11 have DNA sequences inserted into the EcoR1 site andcan be expressed as fusion proteins under the control of the lacpromoter. Therefore they can be screened with antibodies.

In the present case, the method described by Webster et al., 1992(Methods in Molecular Biology vol 10. Immunochemical protocols Ed. M.Manson) were used to screen a λgt11 human pancreas library (Promega).Briefly, the bacterial strain Y1090 was transfected with bacteriophagemixed with molten agarose and plated onto media plates. Theagarose-embedded bacteria grow and make a continuous lawn except wherephage lyse the cells to form clear plaques. At appropriate dilutions ofphage, each discreet plaque arises from one phage infecting onebacterium. Agar plates are then overlayed with a sheet of nitrocellulosemembrane (Protran BA85 0.45 μm, 82 mm, Schleicher and Schuell) that hasbeen soaked in isopropyl β-D-thiogalactoside (IPTG) which induces theβ-galactosidase gene (within the λgt11) into which the cDNA has beeninserted in the unique EcoR1site. If the cDNA is in the correct readingframe and orientation, a fusion protein will be produced which is anextension of the β-galactosidase protein at the carboxyterminus.Membrane-overlayed plates are then incubated at a slightly lowertemperature allowing the production of the fusion protein to increase.The membranes are then removed and washed to remove bacterial debris andprobed with the MoAbs to detect cDNA clones coding for protein sequencesthat react with the antibodies. For this procedure, the membranes werefirst incubated in wash solution (5% milk powder in PBS containing 0.02%Tween (RTM) 20) for 30 minutes to prevent nonspecific binding ofantibody. They were then rinsed in wash solution and placed in petridishes containing neat MoAb and placed on a shaker for 2-3 hours. Theantibody was then removed and the membrane washed in 3 changes of washbuffer for a total of 30 minutes on the shaker. The wash buffer was thenremoved and the membranes were immersed in a suitably diluted horseradish peroxidase labelled anti-mouse antibody (Sigma) for 1 hour on theshaker. The antibody solution was then discarded and the membraneswashed with 3 changes of wash buffer over 30 minutes on a shaker.Antibody binding was then detected using ECL (enhancedchemiluminescence) reagents (Amersham Life Sciences). Equal volumes ofthe 2 reagents were mixed and overlayed on the protein side of themembrane for 1 minute. The excess detection reagent was then drained andthe membranes covered in Saran wrap and exposed to autoradiography film(Hyperfilm™-ECL) in a cassette. The films were developed and matched tothe agar plates containing the plaques. Positive plaques were picked outusing pasteur pipettes and transferred to 0.5 ml of phage eluant(SM:0.1M NaCl, 0.01M MgSO₄.7H₂O, 0.05M Tris base, 0.01%w/v gelatin(swine skin Type 1, adjusted to pH 7.5) containing 50 μl chloroform aspreservative. Positive plaques were rescreened until all plaques on themembrane were positive.

Polymerase Chain Reaction (PCR) Amplification of cDNA Clones

Eight cDNA clones, plaque purified as described previously, wereamplified using the following amplification mix: Taq Plus (Stratagene)10×Low Salt Reaction Buffer 5 μl

dNTPs (Pharmacia) each 200 μM

Forward and Reverse primers each 25 pM (Forward: GTA GAC CCA AGC TTT CCTGGA GCA TGT CAG TAT AGG AGG (SEQ ID NO:5); Reverse: CTG CTC GAG CGG CCGCAT GCT AGC GAC CGG CGC TCA GCT GG (SEQ ID NO:6); Perkin Elmer) Taq PlusDNA polymerase (Stratagene) 1 Unit; cDNA template 2 μl; dH₂O up to 50μl.

The DNA polymerase was added during a 7 minute pre-run at 94° C., i.e.hot start. The reaction mix was overlayed with 100 μl mineral oil. Tubeswere placed in a DNA Thermal Cycler (Perkin-Elmer Cetus, Emeryville,Calif.) programmed as follows:

94° C. (denaturing) 1 min, 55° C. (annealing) 2 min, 72° C. (extension)3 min, for 36 cycles. The last extension was 7 minutes. The PCR productswere stored at 4° C. until analysis.

Analysis of PCR Products

A 1% agarose gel containing 0.5 μg/ml ethidium bromide was prepared inTAE buffer (Tris base 242 g., glacial acetic acid 57.1 ml, 0.5 M EDTA(pH 8.0) 100 ml, dH₂O up to 1000 ml). Ten μl of each PCR product wasloaded with 2 μl of sample buffer. Two μl of 100 base pair and 1 kb DNAladder (Gibco, BRL) were also loaded on either side of the PCR productsfor reference. The gels were run at 100 V for 1 hour. The PCR productswere visualised under UV light and photographed using Polaroid (RTM) 667film (Polaroid, St. Albans, UK)

DNA Sequencing

The identity of the PCR products were checked by sequencing using ABIPRISM Dye Terminator Cycle Sequencing ready reaction kit and ABI 373ASequencer (Applied Biosystems, Perkin-Elmer, Foster City, Calif.).

The cycle sequencing reaction mix was as follows:

terminator ready reaction mix—8 μl, PCR products (10 to 30 ng./μl) 3-6μl, primer 3.2 pM, dH₂O up to 20 μl overlayed with 50 μl light mineraloil. The tubes were placed in the DNA Thermal Cycler and run accordingto the following program: 96° C. for 30 seconds, 50° C. for 15 seconds,60° C. for 4 minutes repeated for 25 cycles. The 20 μl extensionproducts were transferred to 1.5 ml microcentrifuge tubes and 2 l of 3Msodium acetate (pH 4.6) and 50 μl of 95% ethanol were added. The tubeswere vortexed and placed on ice for 10 minutes, then centrifuged at13,000 rpm for 15-30 minutes. The ethanol solution was discarded and thepellet washed in 75% ethanol. The tubes were respun and the ethanolsolution was carefully removed and the pellet dried in a vacuumcentrifuge.

Preparation and Loading of Samples

The dried sample pellets were resuspended in 6 μl of loading buffer(deionized formamide 5 volumes; 50 mg/ml blue dextran in 25 mM EDTA (pH8.0) 1 volume). The samples were vortexed and centrifuged. They werethen heated at 90° C. for 2 minutes and kept in ice until ready to load.The samples were loaded on to a 6% acrylamide gel pre-run for 30 minutesat 1500-2000 V. After loading they were electrophoresed at 2000V for 12hours. The sequence data were analysed by computer.

Eight cDNA clones were purified and sequenced. As discussed above,clones 1.1, 1.2 and 1.3 were found to code for a secretogranin 1 likeprotein; clones 3.1, 4.1 and 5.1 coded for a 67 kd laminin receptor-likeprotein; clone 5.2 coded for a new molecule that has been named ESRP1(endocrine secretion regulatory protein 1. The nucleotide sequence ofESRP1 is given in FIG. 6, and the predicted amino acid sequence that itencodes is shown in FIG. 7.

Cloning of cDNAs into a Eukaryotic Expression Vector (pCR™3-Uni,Invitrogen)

This was done using the unidirectional eukaryotic TA cloning kit(Invitrogen). The linearised vector pCR™3-Uni_does not have a5′-phosphate group on the left arm and therefore will only ligate PCRproducts with a 5′-phosphate. The forward primer used in theamplification of the cDNA was therefore phosphorylated prior to theligation reaction as follows: Forward PCR primer (50-100 μM) 0.5-1 μg,10×-kinase buffer 1 μl, 10 mM ATP 1 μl, sterile water to 9 μl, T4polynucleotide kinase (10 Units/μl) 1 μl were gently mixed in a sterile0.5 ml microcentrifuge tube and incubated at 37° C. for 30-40 minutesand at 94° C. for 5 minutes and then placed on ice. The phosphorylatedforward primer was then used immediately to make a PCR product asdescribed previously and 10 μl of PCR product was analysed on an agarosegel.

The ligation reaction was set up as follows: fresh PCR product(approximately 10 ng) 0.5-1.0 μl, sterile water 5.0-5.5 μl, 10×ligationbuffer 1 μl, pCR™ 3-Uni vector (60 ng) 2 μl, T4 DNA ligase 1 μl. Themixture was incubated at 14° C. for 4 hours or overnight.

The ligation reactions were transformed into Top 10F′ cells (One Shot).One shot cells were thawed on ice and 2 μl of 0.5M β-mercaptoethanol wasadded to the vial. The cells were mixed with 1-2 μl of the ligationreaction and incubated on ice for 30 minutes. The cells were then heatshocked at 42° C. for exactly 30 seconds. SOC medium, 450 μl was thenadded to the vials. They were then incubated on their side at 37° C. for1 hour at 225 rpm in an incubator. Transformed cells were plated on LBplates with ampicillin and incubated overnight at 37° C. Transformantswere picked and cultured for the isolation of plasmids.

Plasmid Purification

Transformed Top 10F′ cells were cultured in LB broth containingampicillin and plasmid DNA was purified using Wizard Miniprep (Promega)kits or the Endotoxin Free Plasmid Kit (Qiagen) for ultra pure DNA.Plasmid DNA was analysed for presence and orientation of insert by PCRand sequencing.

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7 1 1231 DNA Homo sapiens CDS (2)..(1231) 1 g caa ttc cgg gat gaa cagggc ccc atc cgc tgc aac acc aca gtc tgc 49 Gln Phe Arg Asp Glu Gln GlyPro Ile Arg Cys Asn Thr Thr Val Cys 1 5 10 15 ctg ggc aaa atc ggc tcctac ctc agt gct agc acc aga cac agg gtc 97 Leu Gly Lys Ile Gly Ser TyrLeu Ser Ala Ser Thr Arg His Arg Val 20 25 30 ctt acc tct gcc ttc agc cgagcc act agg gac ccg ttt gca ccg tcc 145 Leu Thr Ser Ala Phe Ser Arg AlaThr Arg Asp Pro Phe Ala Pro Ser 35 40 45 cgg gtt gcg ggt gtc ctg ggc tttgct gcc acc cac aac ctc tac tca 193 Arg Val Ala Gly Val Leu Gly Phe AlaAla Thr His Asn Leu Tyr Ser 50 55 60 atg aac gac tgt gcc cag aag atc ctgcct gtg ctc tgc ggt ctc act 241 Met Asn Asp Cys Ala Gln Lys Ile Leu ProVal Leu Cys Gly Leu Thr 65 70 75 80 gta gat cct gag aaa tcc gtg cga gaccag gcc ttc aag gcc att cgg 289 Val Asp Pro Glu Lys Ser Val Arg Asp GlnAla Phe Lys Ala Ile Arg 85 90 95 agc ttc ctg tcc aaa ttg gag tct gtg tcggag gac ccg acc cag ctg 337 Ser Phe Leu Ser Lys Leu Glu Ser Val Ser GluAsp Pro Thr Gln Leu 100 105 110 gag gaa gtg gag aag gat gtc cat gca gcctcc agc cct ggc atg gga 385 Glu Glu Val Glu Lys Asp Val His Ala Ala SerSer Pro Gly Met Gly 115 120 125 gga gcc gca gct agc tgg gca ggc tgg gccgtg acc ggg gtc tcc tca 433 Gly Ala Ala Ala Ser Trp Ala Gly Trp Ala ValThr Gly Val Ser Ser 130 135 140 ctc acc tcc aag ctg atc cgt tcg cac ccaacc act gcc cca aca gaa 481 Leu Thr Ser Lys Leu Ile Arg Ser His Pro ThrThr Ala Pro Thr Glu 145 150 155 160 acc aac att ccc caa aga ccc acg cctgaa gtt cct gcc cca gcc ccc 529 Thr Asn Ile Pro Gln Arg Pro Thr Pro GluVal Pro Ala Pro Ala Pro 165 170 175 acc cct gtt cct gcc acc cct aca acctca ggc cac tgg gag acg cag 577 Thr Pro Val Pro Ala Thr Pro Thr Thr SerGly His Trp Glu Thr Gln 180 185 190 gag gag gac aag gac aca gca gaa gacagc agc act gct gac aga tgg 625 Glu Glu Asp Lys Asp Thr Ala Glu Asp SerSer Thr Ala Asp Arg Trp 195 200 205 gac gac gaa gac tgg ggc agc ctg gagcag gag gcc gag tct gtg ctg 673 Asp Asp Glu Asp Trp Gly Ser Leu Glu GlnGlu Ala Glu Ser Val Leu 210 215 220 gcc cag cag gac gac tgg agc acc gggggc caa gtg agc cgt gct agt 721 Ala Gln Gln Asp Asp Trp Ser Thr Gly GlyGln Val Ser Arg Ala Ser 225 230 235 240 cag gtc agc aac tcc gac cac aaatcc tcc aaa tcc cca gag tcc gac 769 Gln Val Ser Asn Ser Asp His Lys SerSer Lys Ser Pro Glu Ser Asp 245 250 255 ttg gag caa ctg gga agc tta agggtc ctt gga aca cgg ctg gcc agc 817 Leu Glu Gln Leu Gly Ser Leu Arg ValLeu Gly Thr Arg Leu Ala Ser 260 265 270 gag tat aac tgg ggt tgc cca gagtcc agc gac aag ggc gac ccc ttc 865 Glu Tyr Asn Trp Gly Cys Pro Glu SerSer Asp Lys Gly Asp Pro Phe 275 280 285 gct acc ctg tct gca cgt tcc agcacc cag ccg agg cca gac tct tgg 913 Ala Thr Leu Ser Ala Arg Ser Ser ThrGln Pro Arg Pro Asp Ser Trp 290 295 300 ggt gag gac aac tgg gag ggc ctcgag act gac agt cga cag gtc aag 961 Gly Glu Asp Asn Trp Glu Gly Leu GluThr Asp Ser Arg Gln Val Lys 305 310 315 320 gct gag ctg gcc cgg aag aagcgc gag gag cgg cgg cgg gag atg gag 1009 Ala Glu Leu Ala Arg Lys Lys ArgGlu Glu Arg Arg Arg Glu Met Glu 325 330 335 gcc aaa cgc gcc gag agg aaggtg gcc aag ggc ccc atg aag ctg gga 1057 Ala Lys Arg Ala Glu Arg Lys ValAla Lys Gly Pro Met Lys Leu Gly 340 345 350 gcc cgg aag ctg gat gaa ccgtgg cgg tgg ccc ttc ccg gct gcg gag 1105 Ala Arg Lys Leu Asp Glu Pro TrpArg Trp Pro Phe Pro Ala Ala Glu 355 360 365 agc ccg ccc cac aga tgt atttat tgt aca aac cat gtg agg ccg gcc 1153 Ser Pro Pro His Arg Cys Ile TyrCys Thr Asn His Val Arg Pro Ala 370 375 380 ggc cca gcc agg cca ttc acgtgt aca taa tca gag cca caa taa att 1201 Gly Pro Ala Arg Pro Phe Thr CysThr Ser Glu Pro Gln Ile 385 390 395 tta ttt cac aaa aaa aaa acc gga atggcc 1231 Leu Phe His Lys Lys Lys Thr Gly Met Ala 400 405 2 393 PRT Homosapiens Novel Sequence 2 Gln Phe Arg Asp Glu Gln Gly Pro Ile Arg Cys AsnThr Thr Val Cys 1 5 10 15 Leu Gly Lys Ile Gly Ser Tyr Leu Ser Ala SerThr Arg His Arg Val 20 25 30 Leu Thr Ser Ala Phe Ser Arg Ala Thr Arg AspPro Phe Ala Pro Ser 35 40 45 Arg Val Ala Gly Val Leu Gly Phe Ala Ala ThrHis Asn Leu Tyr Ser 50 55 60 Met Asn Asp Cys Ala Gln Lys Ile Leu Pro ValLeu Cys Gly Leu Thr 65 70 75 80 Val Asp Pro Glu Lys Ser Val Arg Asp GlnAla Phe Lys Ala Ile Arg 85 90 95 Ser Phe Leu Ser Lys Leu Glu Ser Val SerGlu Asp Pro Thr Gln Leu 100 105 110 Glu Glu Val Glu Lys Asp Val His AlaAla Ser Ser Pro Gly Met Gly 115 120 125 Gly Ala Ala Ala Ser Trp Ala GlyTrp Ala Val Thr Gly Val Ser Ser 130 135 140 Leu Thr Ser Lys Leu Ile ArgSer His Pro Thr Thr Ala Pro Thr Glu 145 150 155 160 Thr Asn Ile Pro GlnArg Pro Thr Pro Glu Val Pro Ala Pro Ala Pro 165 170 175 Thr Pro Val ProAla Thr Pro Thr Thr Ser Gly His Trp Glu Thr Gln 180 185 190 Glu Glu AspLys Asp Thr Ala Glu Asp Ser Ser Thr Ala Asp Arg Trp 195 200 205 Asp AspGlu Asp Trp Gly Ser Leu Glu Gln Glu Ala Glu Ser Val Leu 210 215 220 AlaGln Gln Asp Asp Trp Ser Thr Gly Gly Gln Val Ser Arg Ala Ser 225 230 235240 Gln Val Ser Asn Ser Asp His Lys Ser Ser Lys Ser Pro Glu Ser Asp 245250 255 Leu Glu Gln Leu Gly Ser Leu Arg Val Leu Gly Thr Arg Leu Ala Ser260 265 270 Glu Tyr Asn Trp Gly Cys Pro Glu Ser Ser Asp Lys Gly Asp ProPhe 275 280 285 Ala Thr Leu Ser Ala Arg Ser Ser Thr Gln Pro Arg Pro AspSer Trp 290 295 300 Gly Glu Asp Asn Trp Glu Gly Leu Glu Thr Asp Ser ArgGln Val Lys 305 310 315 320 Ala Glu Leu Ala Arg Lys Lys Arg Glu Glu ArgArg Arg Glu Met Glu 325 330 335 Ala Lys Arg Ala Glu Arg Lys Val Ala LysGly Pro Met Lys Leu Gly 340 345 350 Ala Arg Lys Leu Asp Glu Pro Trp ArgTrp Pro Phe Pro Ala Ala Glu 355 360 365 Ser Pro Pro His Arg Cys Ile TyrCys Thr Asn His Val Arg Pro Ala 370 375 380 Gly Pro Ala Arg Pro Phe ThrCys Thr 385 390 3 4 PRT Homo sapiens Novel Sequence 3 Ser Glu Pro Gln 14 11 PRT Homo sapiens Novel Sequence 4 Ile Leu Phe His Lys Lys Lys ThrGly Met Ala 1 5 10 5 39 DNA Artificial Sequence Oligonucleotide primer 5gtagacccaa gctttcctgg agcatgtcag tataggagg 39 6 41 DNA ArtificialSequence Oligonucleotide primer 6 ctgctcgagc ggccgcatgc tagcgaccggcgctcagctg g 41 7 6 PRT Artificial Sequence Synthetic peptide 7 Val GlyVal Ala Pro Gly 1 5

What is claimed is:
 1. An isolated antibody or fragment thereof whichspecifically binds to both an anti-T cell receptor (TCR) Vβ antibody anda glycosyl phosphatidyl inositol (GPI) linkage epitope.
 2. The antibodyor fragment thereof according to claim 1, wherein the glycosylphosphatidyl inositol linkage epitope is a glycosyl phosphatidylinositol linked TCR Vβ chain.
 3. The antibody or fragment thereofaccording to claim 1, which further specifically binds to at least onemember selected from the group consisting of a phospholipid, aphospholipid glycan, a single stranded DNA, and a double stranded DNA.4. The antibody or fragment thereof according to claim 1, which furtherspecifically binds to at least one member selected from the groupconsisting of a phosphatidyl inositol, phosphatidyl serine, phospholipidglycan, cardiolipin (diacyl glycerol), single stranded DNA, doublestranded DNA, human pancreatic islet cell, follicular cells of thethyroid, cells of the adrenal medulla, stomach and intestinal tract,salivary glands, striated muscle, and connective tissue.
 5. The antibodyor fragment according to claim 1, which is a monoclonal antibody.
 6. Theantibody or fragment according to claim 1, which is of vertebrate orinvertebrate origin.
 7. The antibody or fragment according to claim 1,which is derived from B cells immortalized by Epstein-Barr virustransformation.
 8. The antibody or fragment according to claim 1, whichis derived from B cells obtained from healthy or diseased humans oranimals.
 9. The antibody or fragment according to claim 1, which isisolated by passing body fluid from an animal down an antigen conjugatedcolumn.
 10. The antibody or fragment according to claim 1, furthercomprising an effector or reporter molecule.
 11. The antibody orfragment according to claim 1, wherein the effector or reporter moleculeis selected from the group consisting of an enzyme, an indicatorcompound, a drug, a toxin and a radioactive label.
 12. The antibody orfragment according to claim 1, which is bound to a biological orsynthetic substance.
 13. A composition comprising the antibody orfragment according to claim 1 together with a pharmaceuticallyacceptable carrier.